JP2023504477A - Biomagnetic microspheres and methods of making and using the same - Google Patents

Abstract

The present invention provides biomagnetic microspheres comprising a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body comprises at least one polymer having a linear backbone and branched chains. having a seed, one end of the linear backbone anchored to the outer surface of the magnetic microsphere body, the other end of the polymer free from the outer surface of the magnetic microsphere body, and a branched chain end of the polymer of the biomagnetic microsphere; is linked to biotin. Additionally, improvements to the biomagnetic microspheres, methods of manufacture and uses are provided. The biomagnetic microspheres are easy to manipulate and use, can be rapidly dispersed in solution and rapidly aggregated, do not require the use of large laboratory equipment such as high-speed centrifuges, are versatile, and can be biotin-mediated. can be linked to selective purification media (e.g., avidin, affinity proteins, polypeptide/protein tags, etc.) and, generally, on a large scale, proteins (including but not limited to antibody-based substances), etc. It can be used for separation and purification of target substances.

Description

This application claims priority from Chinese Patent Application No. 201911209156X with filing date of November 30, 2019 and Chinese Patent Application No. 2020103469030 with filing date of April 28, 2020. The present application cites the full text of the above Chinese patent application.

TECHNICAL FIELD The present invention belongs to the technical field of biochemistry, and specifically relates to biomagnetic microspheres and methods for their preparation and use.

Separation and purification of proteins (including but not limited to antibodies, antibody fragments or fusion proteins thereof) is an important downstream step in the biopharmaceutical manufacturing process, and the efficiency and efficiency of separation and purification affect the quality and production cost of protein drugs. directly affect. Materials such as agarose gels are commonly used as purification columns and purification microsphere carriers for protein purification. In the prior art, the separation and purification of antibody-based substances (e.g., antibody molecules, antibody fragments, or fusion proteins thereof) are mainly performed by production engineers using protein A (abbreviation: Protein A) affinity adsorption columns to extract antibodies in fermentation solutions or reaction solutions. Separate and purify molecules. In the protein A column, the protein A immobilized on the carrier specifically binds to the specific site at the Fc end of the antibody molecule, thereby realizing specific and efficient separation of the antibody from the solution. Materials such as agarose gel are also mainly used as carriers in protein A columns that are generally used at present.

The three-dimensional porous structure of the gel-based material is advantageous in increasing the specific surface area of the material, thereby increasing the sites that can bind purification media (e.g., immobilized protein A) and target proteins (including antibodies). increase the amount of specific binding to The three-dimensional porous structure of the carrier material can greatly improve the number of protein (including antibody) binding sites, but the porous structure inside the carrier increases the retention time of the protein when the protein elutes, and the carrier Some discontinuous spaces or blind spots inside prevent proteins from eluting from inside the material and increase the retention rate. Immobilization of the protein-binding site only on the outer surface of the carrier can avoid the protein product from entering the interior of the material and can greatly reduce the retention time and retention rate of the protein during elution. If only the outer surface of the carrier is used, the specific surface area of the carrier is greatly reduced, the number of protein binding sites is greatly reduced, and the purification efficiency is reduced.

Polymers are macromolecular compounds and can be formed by polymerizing monomer molecules. Polymerization is carried out using monomer molecules with active sites, and the polymerized product can contain a large number of active sites, greatly increasing the number of active sites, and these active sites provide additional corresponding binding sites. can be formed or introduced. Polymers have various types and structures, and have a network structure formed by cross-linking molecular chains with each other, have a linear structure of a single linear molecular chain, and have a branched structure with a plurality of branched chains (e.g. structures such as branched, tree-like, comb-like, and hyperbranched structures), and polymers with different structural types are widely used in different fields, respectively.

In the prior art, purification columns used for protein separation and purification mainly use a covalent bond method to immobilize purification media. Protein A columns for purifying antibody-based substances using protein A as a purification medium are examples. Protein A columns used for separating and purifying antibodies, antibody fragments, fusion proteins thereof, etc., mainly employ a covalent bonding method. is used to immobilize protein A, and the C-terminal cysteine covalently binds protein A to the carrier. Although the covalent attachment method can ensure that the purification medium (e.g. protein A) is firmly fixed to the support, after multiple uses of the purification column (e.g. protein A column), the purification medium (e.g. protein A) The binding performance of is reduced and the purification effect is reduced. Therefore, in order to ensure relatively high purification efficiency and quality, the operator needs to replace all the fillers in the affinity chromatography column. It is labor and time consuming and causes high purification costs.

The present invention provides biomagnetic microspheres, which can be used to separate and purify target substances, particularly protein-based substances (including but not limited to antibody-based proteins), and can bind target substances in high throughput. Not only can the retention rate of target substances during elution be effectively reduced, purification media (e.g. affinity proteins) can be conveniently exchanged, and it is fast, high-throughput, reusable, and reusable. and can greatly reduce the purification cost of the target substance. For example, when affinity proteins are used as purification media, purification costs for antibody-based proteins can be greatly reduced.

1. A first aspect of the present invention provides biomagnetic microspheres, said biomagnetic microspheres comprising a magnetic microsphere body, the outer surface of said magnetic microsphere body having at least one linear backbone and a polymer having a branched chain, one end of the linear main chain is fixed to the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the biomagnetic microsphere Biotin or biotin analogues are linked to the branched ends of the polymer. Said biotin or biotin analogue can not only serve as a purification medium, but can also be linked as a linking element to still other types of purification media.

Said biomagnetic microspheres are also called biotin magnetic microspheres or biotin magnetic beads.

The term "anchored to" refers to the linear backbone being "anchored to" the outer surface of the magnetic microsphere body in a covalent manner.

In addition, the linear main chain is covalently fixed to the outer surface of the magnetic microsphere body in a direct manner, or shared on the outer surface of the magnetic microsphere body in an indirect manner via a linking group (linking element). Cohesively fixed.

Furthermore, the number of branched chains in the polymer is plural, preferably at least three.
In one preferred form, the size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 600 μm, 600 μm, 650 μm, Any one particle size scale of 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm or a range between any two particle size scales, said diameter size being an average value.

In one preferred form, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.2 to 6 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.4 to 5 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.5 to 3 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.2 to 1 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.5 to 1 μm.

In one preferred form, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In one preferred form, the magnetic microsphere body has a diameter of 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, The deviation is ±20%, more preferably ±10%.

In one preferred form, the backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone. See the "Nouns and Terminology" section for definitions of said acrylic polymers. In one preferred form, the polyolefin backbone is at the same time an acrylic polymer backbone (ie the linear backbone of the polymer is both a polyolefin backbone and provided from an acrylic polymer backbone).

In a preferred embodiment, the monomer unit of the acrylic polymer is preferably any one of acrylic monomer molecules such as acrylic acid, acrylate, acrylic acid ester, methacrylic acid, methacrylate, and methacrylic acid ester, or selected from a combination thereof. Said acrylic polymer may be obtained by polymerizing any of the above monomers, or may be obtained by copolymerizing the corresponding combination of the above monomers.

In one preferred form, the branched chains of the polymer are covalently attached to biotin or biotin analogues by functional group-based covalent bonds, covalently attaching biotin or biotin analogues to the branched ends of the polymer. It can be obtained by covalent reaction of biotin or biotin analogues with functional groups contained in branched chains of polymer molecules on the outer surface of biomagnetic microspheres. Here, one preferred embodiment of said functional group is a specific binding site (see "Nouns and Terms" section of specific embodiments for definition).

The covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group. Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. A preferred salt form of the carboxyl group is a sodium salt form, for example, COONa, and a preferred salt form of the amino group may be an inorganic salt form or an organic salt form. Often include, but are not limited to, hydrochloride, hydrofluoride, and like forms. The "combination of functional groups" refers to all branched chains of all polymer molecules on the outer surface of one magnetic microsphere, which can form covalent bonds based on the involvement of different functional groups. Taking biotin as an example, all biotin molecules on the outer surface of one biotin magnetic microsphere can covalently bond with different functional groups, but one biotin molecule can only link with one functional group. .

In one preferred form, the linear backbone of the polymer is covalently attached to the outer surface of the magnetic microsphere body either directly or indirectly via a linking group.

In one preferred form, said magnetic microsphere body is a magnetic material coated with _SiO2 . Alternatively, _SiO2 may itself be a silane coupling agent with active sites.

In one preferred form, the magnetic material is selected from iron oxides, iron compounds, iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, or a combination thereof.

Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNiMo), FeSi , FeAl, _FeSiAl , _MO.6Fe2O3 , GdO, or a combination thereof, wherein Re is a rare earth element and M is Ba, Sr, Pb, i.e., _{The MO.6Fe2O3} is _{BaO.6Fe2O3 , SrO.6Fe2O3 or PbO.6Fe2O3 .}

2. A second aspect of the present invention provides biomagnetic microspheres, and based on said biomagnetic microspheres provided in the first aspect of the present invention, said biotin or biotin analogue is further added as a linking element to a purification medium. are connected. That is, the branched ends of the polymer are linked to the purification medium via a linking element, and the linking element comprises the biotin or biotin analogue.

The purification media may contain, but are not limited to, avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and forms an affinity complex binding action with the avidin.

The avidin may be, but is not limited to, streptavidin, modified streptavidin, streptavidin analogs, or combinations thereof.

In a preferred embodiment, the polypeptide tag includes a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, and a variant sequence of WRHPQFGG. tags, tags comprising RKAAVSHW sequences, tags comprising variant sequences of RKAAVSHW, or combinations thereof, or variants thereof. The Streg tags include WSHPQFEK and variants thereof.

In one preferred form, said protein type tag is selected from affinity proteins, SUMO tags, GST tags, MBP tags and any combination thereof or variants thereof.

In one preferred form, the affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

In a preferred embodiment, the antibody-based tag is an antibody, antibody fragment, antibody single chain, single chain fragment, antibody fusion protein, antibody fragment fusion protein, derivative of any, or any of is a variant of

In one preferred form, the antibody-based tag is an anti-protein antibody.
In one preferred form, the antibody-based tag is an anti-fluorescent protein antibody.
In one preferred form, the antibody-based tag is a nanobody.
In one preferred form, the antibody-based tag is an anti-protein nanobody.
In one preferred form, the antibody-based tag is an anti-fluorescent protein nanobody.
In one preferred form, the antibody-based tag is an anti-green fluorescent protein or variant nanobody.
In one preferred form, the antibody-based tag is an Fc fragment.

Modes of linkage between the purification medium and the biotin or biotin analog include, but are not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or combinations thereof.

In one preferred form, the covalent bond is a dynamic covalent bond, more preferably the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In one preferred form, said supramolecular interactions are coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions, or combinations thereof.

In one preferred form of the biomagnetic microspheres, the purification media are linked to the branched ends of the polymer by linking elements comprising affinity complexes.

In one preferred form, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In one preferred form, said biotin or biotin analogue is bound to avidin or an avidin analogue by affinity complexing and said purification medium is bound directly or indirectly to said avidin or avidin analogue.

In one preferred form, the purification medium is linked to biotin or a biotin analogue at the branched end of the polymer by an avidin-type tag-purification medium covalent complex, and is linked to the biotin or biotin analogue by an avidin-type tag. form the linking member of the affinity complex, and in a further preferred form, the purification medium binds biotin or a biotin analog at the branched chain end of the polymer to the affinity complex by means of an avidin-purification medium covalent complex. Form a connecting element.

3. A third aspect of the invention provides biomagnetic microspheres, based on said biomagnetic microspheres provided by the first aspect of the invention, further comprising said biotin or biotin analogue as a linking member, further comprising: It is linked to avidin or an avidin analogue by an affinity complex binding activity.

Said biomagnetic microspheres are also called avidin magnetic microspheres or avidin magnetic beads.

Said avidin or avidin analogue can not only serve as a purification medium, but can also be linked with other types of purification media as a linking element. An affinity complex binding is now formed between said biotin or biotin analogue and said avidin or avidin analogue.

In one preferred form, based on said biomagnetic microspheres provided in the first aspect of the invention, it further comprises avidin binding to said biotin. An affinity complex binding is now formed between biotin and avidin. That is, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and forms an affinity complex with the avidin.

In one preferred form, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof.

4. A fourth aspect of the invention provides biomagnetic microspheres, based on said biomagnetic microspheres provided in the third aspect of the invention, further comprising affinity linked to said avidin or avidin analogue. Contains protein. At this time, biotin or a biotin analogue and avidin or an avidin analogue form a binding action of an affinity complex between them as linking elements, and the affinity protein can not only serve as a purification medium, It can also be a connecting element, preferably a purification medium.

Said biomagnetic microspheres are also called affinity protein magnetic microspheres or affinity protein magnetic beads.

In one preferred form, based on said biomagnetic microspheres provided in the second aspect of the present invention, said purification medium is an affinity protein, said biomagnetic microspheres further comprising avidin linked to said affinity protein. and biotin attached to said avidin, wherein said biotin is linked to said branch of said polymer, wherein said purification medium is linked to said branch of said polymer by a linking element, and contains the affinity complex formed by biotin and avidin.

In a preferred embodiment, the affinity protein is any one of protein A, protein G, protein L, or denatured proteins thereof.

5. A fifth aspect of the invention provides a method (see FIG. 3) of manufacturing said biomagnetic microspheres provided in the first aspect of the invention, said method comprising the following steps.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microspheres A, and the magnetic microsphere body is made of _SiO2 . In the case of coated magnetic materials, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. Said silane coupling agent is preferably an aminated silane coupling agent.

(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , to form magnetic microspheres B containing carbon-carbon double bonds.

(3) Polymerizing an acrylic monomer molecule (for example, sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer has a linear main chain. and a branched chain containing a functional group, the polymer is covalently bonded to the outer surface of the magnetic microsphere B by one end of the linear backbone to form the acrylic polymer-modified magnetic microsphere C.

See the "Nouns and Terminology" section for definitions of the functional groups of the acrylic monomer molecules and polymer branches.

Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a formate, ammonium, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. The "combination of functional groups" refers to functional groups contained in all branched chains of all polymers on the outer surface of one magnetic microsphere, and the types thereof may be one or more. . It matches the meaning of "combination of functional groups" defined in the first mode.

Also preferably, said functional group is a specific binding site.

(4) biomagnetic microspheres D attached to biotin or biotin analogues by covalently coupling biotin or biotin analogues to the branched ends of the polymer via functional groups contained in the branched chains of the polymer; (biotin magnetic microspheres) are obtained.

6. A sixth aspect of the present invention provides a method of manufacturing said biomagnetic microspheres provided in the second aspect of the present invention, said method comprising the following steps.

(i) providing the biomagnetic microspheres of claim 1, which can be manufactured in steps (1) to (4) of the fifth aspect;

(ii) Linking a purification medium to biotin or a biotin analogue at the branched end of the polymer of said biomagnetic microspheres to obtain biomagnetic microspheres with bound purification medium.

7. A seventh aspect of the present invention provides a method of manufacturing said biomagnetic microspheres provided in the second aspect of the present invention, said method comprising the following steps.

(i) providing the biomagnetic microspheres of claim 1, which can be manufactured in steps (1) to (4) of the fifth aspect;

(ii) a covalent complex of an avidin or avidin analogue with a purification medium (e.g., an avidin-purification medium covalent complex) as a starting material for providing a purification medium, attached to the branched ends of a polymer, said forming an affinity complex binding action between biotin or a biotin analogue and said avidin or avidin analogue to obtain biomagnetic microspheres with a purification medium.

Optionally independently, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing.

Optionally, independently, it includes exchanging the purification medium, which can be achieved by exchanging the covalent complexes of the avidin or avidin analogue with the purification medium.

8. An eighth aspect of the invention provides a method of manufacturing the biomagnetic microspheres provided in the fourth aspect of the invention (see FIG. 3 for an example), the method comprising the following steps.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microspheres A, and the magnetic microsphere body is made of _SiO2 . In the case of coated magnetic materials, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. At this time, the silane coupling agent is preferably an aminated silane coupling agent.

(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , to form magnetic microspheres B containing carbon-carbon double bonds.

(3) Polymerize acrylic monomer molecules (for example, sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer has a linear main chain and The polymer is covalently bonded to the outer surface of the magnetic microsphere B by one end of the linear backbone to form the acrylic polymer-modified magnetic microsphere C.

A preferred embodiment of said functional group is a specific binding site.

Other preferred forms of said functional groups are consistent with the first aspect above.

(4) Biotin is covalently coupled to the functional groups contained in the branched chains of the polymer to obtain biotin-modified biomagnetic microspheres D (biotin magnetic microspheres).

(5) binding the avidin-affinity protein covalent complex E to the branched end of the polymer through the specific binding action of biotin and avidin to form the binding action of the affinity complex between biotin and avidin; , to obtain biomagnetic microspheres F (said biomagnetic microspheres F are affinity protein magnetic microspheres because they bind to affinity proteins).

Independently and optionally, (6) sedimenting the biomagnetic microspheres F with a magnet, removing the liquid phase, and washing.

Further independently optionally, (7) exchanging said avidin-affinity protein covalent complex E.

9. A ninth aspect of the invention provides the use of said biomagnetic microspheres according to the first to fourth aspects of the invention in the separation and purification of proteinaceous substances.

A preferred embodiment is the use of the biomagnetic microspheres in the separation and purification of antibody-based substances.

The antibody-based substances refer to proteins of antibodies and antibody fragments, including, but not limited to, antibodies, antibody fragments, antibody fusion proteins, and antibody fragment fusion proteins.

When the purification medium is linked to the branched ends of the polymer by a linking element comprising an affinity complex, the use further optionally comprises the recycle use of the biomagnetic microspheres, i.e. reuse after exchange of the purification medium. including.

10. A tenth aspect of the invention is the use according to the first to fourth aspects of the invention in the separation and purification of antibody-based substances of biomagnetic microspheres, in particular antibodies, antibody fragments, antibody fusion proteins, antibody fragment fusions. Use in protein separation and purification is provided.

Said purification medium is an affinity protein.

In one preferred form, the affinity protein is linked to the polymer branches in a biotin-avidin-affinity protein fashion.

Where said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex (e.g. biomagnetic microspheres according to the fourth aspect), said use further optionally comprises biomagnetic microspheres. Includes recycling of spheres, ie recycling after exchange of affinity proteins.

11. An eleventh aspect of the present invention provides biomagnetic microspheres. The outer surface of the magnetic microsphere main body has at least one type of polymer having a linear main chain and a branched chain, and one end of the linear main chain is fixed to the outer surface of the magnetic microsphere main body. The other end is free from the outer surface of the magnetic microsphere main body, and a purification medium is linked to the branched chain end of the polymer of the magnetic microsphere, and the purification medium is an avidin-type tag, a polypeptide-type tag, or a protein. A type tag, an antibody type tag, an antigen type tag, or a combination thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

In a preferred embodiment, the polypeptide tag includes a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, and a variant sequence of WRHPQFGG. tags, tags containing RKAAVSHW sequences, tags containing variant sequences of RKAAVSHW, or any combination thereof and variants thereof. The Streg tag includes WSHPQFEK or variants thereof.

In one preferred form, said protein type tag is selected from any of affinity proteins, SUMO tags, GST tags, MBP tags and combinations thereof or variants thereof.

In a preferred embodiment, the outer surface of the main body of the magnetic microsphere has at least one type of polymer having a linear main chain and a branched chain, and one end of the main linear chain is attached to the outer surface of the main body of the magnetic microsphere. Covalently immobilized, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the affinity protein is linked to the branched chain end of the polymer of said magnetic microsphere.

Preferably, there is also a binding function of the affinity complex on the branched backbone between said affinity protein and the linear backbone of the polymer.

More preferably, said affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

The main advantages and positive effects of the invention include the following.

One of the core of the present invention is the structure of the biomagnetic microspheres, in which linear backbone-containing polymer molecules are covalently anchored to the outer surface of the magnetic microsphere body, and these polymer molecules have an additional large number of functions. functionalized branched chain to which purification media (including but not limited to biotin, avidin, affinity proteins, polypeptide tags, protein tags, etc.) are linked. The above structure not only provides a purification medium suspended on the side ends of a large amount of polymeric linear backbones on the outer surface of the biomagnetic microspheres to avoid the high retention rate caused by conventional network structures, but also has a specific surface area of can provide a large number of target substance (eg, antibody) binding sites, overcoming the limitations of . Here, the type of purification medium can be selected according to the type of substance to be purified. In addition, if the purification medium is in the form of an affinity complex and is linked to the branched chains of the polymer by strong non-covalent interactions, a branched backbone between the purification medium (e.g. affinity protein) and the polymer linear backbone There may also be a binding activity of the affinity complex in , whereby the purification medium (eg parent protein) can be readily exchanged. When the target substance to be separated and purified is an antibody-based protein, the purification medium is usually an affinity protein. The description of the manufacturing and principle part of the magnetic microspheres should be combined and understood.

Further, the core of the present invention lies in the construction process (manufacturing method) of the biomagnetic microsphere structure described above, in which a plurality of binding sites are formed on the outer surface of the magnetic beads (the outer surface of the main body of the biomagnetic microsphere) by chemically modifying the outer surface. followed by covalent attachment of a polymer molecule to a single binding site on the outer surface of the magnetic bead, the polymer molecule being covalently attached by one end of the linear backbone to the single binding site on the outer surface of the magnetic bead. , a large number of side-branched chains are distributed along the linear main chain, and the side-branched chains carry the nascent binding sites, thereby multiplying the binding sites by multiples, tens, hundreds, hundreds, or even thousands of times. Furthermore, based on specific purification requirements, specific purification media can be linked to the nascent binding sites of polymer branches to generate corresponding specific target molecules (especially biochemical molecules, antibody-based protein molecules). (including but not limited to) capture. Also, a single binding site of a biomagnetic microsphere can covalently bind not only to one linear polymer backbone, but also to two or more linear backbones. , preferably does not cause chain build-up and increase the retention rate.

Preferably, a single binding site can pull only one linear backbone, thus providing the linear backbone with a large active space.

Also preferably, one binding site pulls only two linear backbones, providing as much activity space for the linear backbones as possible.

The main advantages and positive effects of the present invention further include:
(1) The structural design of the present invention coats the magnetic microsphere surface with a polymer carrying a large amount of branched special structures to overcome the limitation of specific surface area to provide a large amount of purification media binding sites and to The number of purification media that can be bound to the surface of the sphere has been expanded several times, ten times, several tens of times, hundreds of times, hundreds of times, and even a thousand times, and high-throughput binding to target substances has been achieved. The substance is a proteinaceous substance. Biomagnetic microspheres efficiently capture target substances (e.g., target proteins, including but not limited to antibodies, antibody fragments, or fusion proteins thereof) from mixed systems onto the magnetic microspheres for high-throughput binding. to achieve high-throughput separation.

(2) The flexibility of the polymer chain itself can be used, and the polymer chain can flexibly oscillate in the reaction purification mixing system, expanding the activity space of the purification medium and increasing the capture rate and binding amount for proteins. promote rapid and sufficient binding of the target substance, realizing high efficiency and high throughput.

(3) The structural design of the present invention affects the efficiency of purified target substances (e.g., target proteins, including but not limited to antibodies, antibody fragments, or fusion proteins thereof) when biomagnetic microspheres elute. It can achieve effective elution, effectively reduce the retention time and retention rate of the target substance, and achieve high efficiency and high yield. The purification medium can be linked to the branched chain ends of the polymer, and in one aspect, the structure of the polymer does not form a network structure, does not cause accumulation of branches, and avoids the occurrence of discontinuous spaces and blind spots. , avoiding the high residence time and high residence fraction of conventional reticulated structures. On the other hand, the branched chains of the polymer further act as spatial spacing, allowing the purification media to be well distributed in the mixed system, away from the magnetic microsphere surface and the internal backbone of the polymer, increasing the efficiency of target substance capture. In addition, it effectively reduces the retention time and retention rate of the target substance during the subsequent elution step, achieving high throughput, high efficiency and high percentage separation. The structural design of the present invention not only can take advantage of the high flexibility of the linear backbone, but also combines the advantages of high-fold amplification of the number of branches, resulting in high-rate, high-throughput binding, high efficiency, and high ratio. (high yield) separation is better achieved.

(4) Purification media (e.g., affinity proteins) of the biomagnetic microspheres of the present invention are linked to the polymer branched ends on the outer surface of the magnetic beads by a strong non-covalent binding force in the manner of affinity complexes, and the purification media (e.g., When the affinity protein) needs to be renewed or replaced, the purification medium can be easily and quickly eluted from the microspheres and recombined with new purification medium, quickly restoring the purification performance of the magnetic microspheres, Biomagnetic microspheres can be regenerated and used multiple times, thereby reducing separation and purification costs.

(5) The biomagnetic microspheres of the present invention are easy to manipulate and use. When the magnetic microspheres bound to the target substance are separated from the system, it is easy to operate, and the aggregation state and position of the magnetic microspheres can be efficiently manipulated simply by using a small magnet. Realizes high-speed dispersion or high-speed sedimentation to make the separation and purification of target substances (such as antibodies) simple and fast, without the need to use large-scale experimental equipment such as high-speed centrifuges, greatly reducing the cost of separation and purification. Reduce.

(6) The biomagnetic microspheres provided by the present invention are versatile and have selectable purification media. Depending on the type of specific purification substrate, the magnetic microsphere system can be equipped with flexible purification media to achieve the capture of specific target molecules (especially proteinaceous substances). For example, affinity proteins are targeted and generally used for large-scale separation and purification of antibody-based substances, including, but not limited to, antibodies, antibody fragments, or fusion proteins thereof.

1 is a structural schematic diagram of a biomagnetic microsphere provided in the first aspect of the present invention; FIG. Here, the magnetic microsphere body is exemplified by Fe ₃ O ₄ coated with SiO ₂ and biotin is shown as the purification medium. In the figure, the number of polymer molecules (4) is for simplicity and schematic only, and does not mean that the number of polymer molecules on the outer surface of the magnetic microspheres is limited to 4. can be controlled and adjusted based on the content of each raw material in Similarly, the number of branches pendant to the linear main chain side ends is for illustrative purposes only and is not intended to limit the number of side branches in the polymer molecules of the present invention. FIG. 4 is a structural schematic diagram of a biomagnetic microsphere provided in the fourth aspect of the present invention; Protein A is shown as the purification medium and is attached to the branched ends of the brush-like structure by the “biotin-avidin-protein A scheme”. Here, the biomagnetic microsphere body is exemplified by Fe ₃ O ₄ coated with SiO ₂ . In the figure, the number of polymer molecules and the number of branched chains at the end of the main chain are merely examples, and do not limit the number of side branched chains of the polymer molecule of the present invention. Figure 2 is a flow chart of a method for manufacturing biomagnetic microspheres provided in the fourth aspect of the invention; Here, the manufacturing process from the amino group-modified magnetic microsphere A to the biomagnetic microsphere D corresponds to the manufacturing method of the biomagnetic microsphere provided in the fifth aspect. Biomagnetic Microsphere D (biotin magnetic beads) is the result of experiments before and after binding with protein A-eGFP-avidin. It was incubated once with the result of RFU value measurement. Protein A-eGFP-avidin (SPA-eGFP-avidin) was produced by an in vitro protein synthesis system, and the supernatant after IVTT reaction (abbreviated as IVTT supernatant) was obtained, and before binding with biomagnetic microsphere D and compare the changes in solution RFU values. In the two protein A-eGFP-avidins, where the avidin corresponding to number 1 is Streptavidin and the avidin corresponding to number 2 is Tamvavidin2. "Total" corresponds to the IVTT supernatant RFU value before binding (before biomagnetic microsphere treatment) and "Supernatant" corresponds to the IVTT supernatant RFU value after binding (after biomagnetic microsphere treatment). Fig. 2 shows experimental results of producing biomagnetic microspheres F (Protein A magnetic beads). This is the measurement result of the RFU value, and protein A-eGFP-avidin was saturated bound. The biomagnetic microspheres D were repeatedly combined with the solution obtained after the IVTT reaction expressing protein A-eGFP-avidin (abbreviated as IVTT supernatant) to form biomagnetic microspheres saturated with protein A-eGFP-avidin. Get Sphere F. where the avidin corresponding to 1 is Streptavidin, the avidin corresponding to 2 is Tamvavidin2, supernatant corresponds to IVTT supernatant not treated with biomagnetic microspheres, Flow-through1, Flow-through 2 (flow-through 2) and Flow-through 3 (flow-through 3) correspond to 3 cycles of elution (debinding release), followed by continuous incubation of magnetic microspheres with avidin-protein A (capture binding). , flow-throughs 1, 2 and 3 obtained sequentially using the same source and amount of IVTT supernatant each time. Biomagnetic microsphere F is an experimental result of separating and purifying an antibody, and the avidin in biomagnetic microsphere F is Streptavidin. The biomagnetic microspheres F were eluted after incubation with the antibody IgG solution, the antibody IgG was captured and separated from the antibody solution, eluted and released into the eluate, and the corresponding purified antibody-containing eluate was subjected to denaturing polyacrylamide gel electrophoresis. It is a migration (SDS-PAGE) test result. Here, lanes 1, 2, 3, and 4 correspond to antibody elution bands when the column bed volumes of SPA-magnetic microspheres were 2 microliters, 6 microliters, 18 microliters, and 54 microliters, respectively. 5 is an antibody elution band of a positive control, a commercially available Protein A agarose column of Seiko Biotechnology (Shanghai) Co., Ltd. (hereinafter referred to as Seiko) with a column volume of 7.5 microliters. where M corresponds to the Marker molecular weight mark. It is the experimental result of repeated use of biomagnetic microsphere F, and the avidin in biomagnetic microsphere F is Streptavidin. Biomagnetic microspheres F were incubated with an antibody solution (antibody binding), washed, and eluted (antibody release) three times, and the eluate was subjected to an SDS-PAGE electrophoresis test. Here, lanes 1, 2, 3, and 4 correspond to antibody elution bands when the column bed volumes of SPA-magnetic microspheres were 2 microliters, 6 microliters, 18 microliters, and 54 microliters, respectively. 5 is an antibody elution band of a positive control, a commercially available Protein A agarose column of Seiko Biotechnology (Shanghai) Co., Ltd. (hereinafter referred to as Seiko) with a column volume of 7.5 microliters. where M corresponds to the Marker molecular weight mark. Fig. 4 shows the results of regeneration experiments of biomagnetic microspheres, and the results of measurement of fluorescence relative values using protein A-eGFP-Tamvavidin2; Biomagnetic microsphere D was repeatedly bound with the solution obtained after IVTT reaction of protein A-eGFP-avidin to obtain biomagnetic microsphere F (1) saturated with protein A-eGFP-avidin. The Protein A-eGFP-avidin is subsequently eluted from the biomagnetic microspheres D, and the biomagnetic microspheres D are bound a second time with the solution obtained after the IVTT reaction of fresh Protein A-eGFP-avidin,2 A second round of biomagnetic microspheres F (2) saturated with protein A-eGFP-avidin was obtained. The above steps were repeated for the third time to obtain biomagnetic microspheres F(3) saturated with protein A-eGFP-avidin. Here, supernatant corresponds to IVTT supernatant not treated with biomagnetic microspheres, Flow-through1, Flow-through2, Flow-through3 are magnetic Flow-throughs 1, 2, 3 were obtained sequentially, microspheres were successively incubated with avidin-protein A three times, each time using the same IVTT supernatant (taken fresh). Fig. 2 shows experimental results of regenerating biomagnetic microspheres and isolating and purifying antibodies after regenerating. Protein A-eGFP-Tamvavidin2 was employed. Biomagnetic microspheres F saturated with protein A-eGFP-avidin were eluted with denaturing buffer, and the eluate containing the eluted protein A-eGFP-avidin was subjected to SDS-PAGE. Lanes 1, 2 and 3 are eluted after the first saturation binding, eluted after the second saturation binding, and eluted after the third saturation binding, respectively. A band from the eluate containing protein A-eGFP-avidin is shown. Lane 4 shows the purified antibody protein band eluted with the elution buffer after incubating the third renatured Biomagnetic Microsphere F with commercial newborn bovine serum. where M corresponds to the Marker molecular weight mark. Fig. 2 shows test results of the amount of protein G supported by biomagnetic microspheres G (protein G magnetic beads, protein G magnetic beads), and measurement results of RFU values. The IVTT supernatant of biomagnetic microsphere D and protein G-eGFP-avidin was incubated three times to obtain biomagnetic microsphere G saturated bound to protein G-eGFP-avidin. Here, the Supernatant corresponds to the IVTT supernatant not treated with biomagnetic microspheres and the corresponding flow-throughs by three incubations: FT1 (flow-through 1), FT2 (flow-through 2), FT3 (flow-through), respectively. Through 3) was obtained. It is an experimental result of antibody separation and purification using biomagnetic microsphere G (Protein G magnetic beads). The biomagnetic microspheres G were eluted after incubation with the antibody IgG solution, the antibody IgG was captured and separated from the antibody solution, eluted and released into the eluate, and the corresponding purified antibody-containing eluate was subjected to SDS-PAGE test. gone. where M corresponds to the Marker molecular weight mark. Fig. 3 shows the RFU value measurement results of biomagnetic microsphere H (magnetic beads with anti-EGFP nano-antibodies) binding to eGFP protein. Biomagnetic Microsphere H and eGFP protein IVTT supernatant were incubated to bind the eGFP protein. Here, Total corresponds to IVTT supernatant not treated with biomagnetic microspheres and Flow-through corresponds to flow-through incubated once. Fig. 3 shows experimental results of separation and purification of eGFP protein using biomagnetic microsphere H (antiEGFP magnetic beads). Biomagnetic Microsphere H is eluted after incubation with eGFP protein solution, eGFP protein is captured and separated from the stock solution, eluted and released into the eluate, SDS-PAGE study of the eluate containing the corresponding purified eGFP protein. This is the result. where M corresponds to the Marker molecular weight mark.

The following will further describe the present invention with reference to specific embodiments and examples, while referring to the drawings. It should be understood that these examples are only illustrative of the invention and do not limit the scope of the invention. Experimental methods that do not specify specific conditions in the following examples are preferentially based on the conditions guided according to the above specific embodiments, followed by conventional conditions, such as "Sambrook et al., Molecular Cloning: Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free protein synthesis experiment manual", "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis [methods] and .2008” and the conditions suggested by the manufacturer.

Unless otherwise stated, percentages and parts used herein refer to weight percentages and parts by weight.

Unless otherwise stated, all materials and reagents used in the examples of the present invention are commercially available products.

All temperature units in the present invention are degrees Celsius (° C.) unless otherwise specified.

Nucleotide and/or Amino Acid Sequence Listing SEQ ID No: 1, nucleotide sequence of protein A, 873 bases in length.
SEQ ID No: 2, nucleotide sequence of tamavidin2, 423 bases in length.
SEQ ID No: 3, nucleotide sequence of mEGFP, length is 714 bases.
SEQ ID No: 4, nucleotide sequence of protein G antibody binding site, 585 bases in length.
SEQ ID No: 5, amino acid sequence of anti-eGFP antibody, 117 amino acids in length.
SEQ ID No: 6, nucleotide sequence of mScarlet, 693 bases in length.

Nouns and Terms The following is an analysis or explanation of the meaning of "nouns", "terms" partly related to which the present invention employs, in order to better understand the present invention. A corresponding analysis or explanation applies to the full text of the invention, applies below, and applies above. When referring to cited documents in the present invention, the definitions of related terms, nouns, and phrases in the cited documents are also cited. If the definition in the cited document conflicts with the definition in the present invention, it shall not affect the cited component, substance, composition, material, system, formulation, type, method, equipment, etc. according to the content specified in

Magnetic beads: Ferromagnetic or possibly ferromagnetizable microspheres with a small particle size can also be described as magnetic beads, preferably with a diameter size of 0.1 μm to 1000 μm. Examples of magnetic beads of the present invention include magnetic microspheres A, magnetic microspheres B, magnetic microspheres C, biomagnetic microspheres D (biotin magnetic beads), biomagnetic microspheres F (protein A magnetic beads), biomagnetic microspheres Including but not limited to Sphere G (protein G magnetic beads), biomagnetic microsphere H (magnetic beads with antiEGFP nanoantibody), biomagnetic microsphere K (antibody magnetic beads).

Magnetic microsphere body: Magnetic beads with modified sites (magnetic microspheres with sites that can bind). For example, silica-coated magnetic material particles, more specifically aminated silica-coated magnetic material particles.

Magnetic microsphere A: Amino group-modified magnetic microsphere.

Magnetic microsphere B: A magnetic microsphere having a carbon-carbon double bond.

Magnetic Microsphere C: Acrylic polymer-modified magnetic microsphere.

Biotin Beads: Magnetic beads to which biotin or biotin analogues are bound and which are capable of binding specifically to avidin-type tagged substances. Its advantage is that the target protein can be labeled with avidin or a protein mutant of avidin and then integratedly expressed in the form of a fusion protein, which is convenient to use. Also called biotin microspheres. The biotin or biotin analogue herein can be a purification medium and can also be a linking element.

Biomagnetic Microspheres D: Magnetic microspheres bound with biotin or biotin analogues, biotin magnetic beads. Biotin herein can be a purification medium and can also be a linking element.

Avidin Beads: Magnetic beads to which avidin or an avidin analogue is bound. It can specifically bind to biotin-tagged substances. Also called avidin magnetic microspheres.

Biomagnetic microspheres F: Magnetic microspheres bound with protein A, protein A magnetic beads. A biotin-modified biomagnetic microsphere D and an avidin-protein A covalent complex E are bound to obtain.

Affinity protein magnetic beads: Magnetic microspheres bound with affinity proteins, which can be used for the separation and purification of antibody-like substances. Also called affinity protein microspheres.

Biomagnetic Microspheres G: Magnetic microspheres bound with Protein G, Protein G magnetic beads. For example, biotin-modified biomagnetic microspheres D and avidin-protein G covalent complexes are bound together.

Biomagnetic Microsphere K: Magnetic beads with attached antibody-type tags. It can be used for separation and purification of target substances that can specifically bind thereto. Also called antibody magnetic microspheres or antibody magnetic beads.

Nano-antibody magnetic beads: magnetic beads with attached nano-antibodies. It can be used for separation and purification of target substances that can specifically bind thereto. Also called nano-antibody magnetic microspheres.

Biomagnetic microspheres H: nanoantibody magnetic beads, magnetic microspheres bound with nanoantibody antiEGFP (antiEGFP magnetic beads). Avidin-antiEGFP covalent complex is bound to obtain.

Polymer: in the present invention broadly includes oligomers and macromolecules, having at least 3 structural units or having a molecular weight of at least 500 Da (said molecular weight is defined by any suitable characterization scheme, e.g. number average molecular weight, weight average molecular weight, viscosity average molecular weight can be employed).

Polyolefin chain: refers to a polymer chain containing no heteroatoms covalently bonded only by carbon atoms. In the present invention, a polyolefin backbone in a comb-like structure, such as the linear backbone of an acrylic polymer, is primarily concerned.

Acrylic polymer: Refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure, the copolymerization form of the copolymer is not particularly limited, and a linear main chain and weighed side groups COO- and the linear backbone of the acrylic polymer may contain heteroatoms. Here, the carbon-carbon double bond may have other substituents such as methyl substituents (corresponding to —CH ₃ C(COO—)—C—) as long as they do not hinder the progress of the polymerization reaction. may Here, COO- may exist in the form of -COOH, may be in the form of a salt (eg sodium salt), or in the form of a formate (preferably a formate alkyl ester, such as methyl formate -COOCH ₃ , ethyl formate-COOCH ₂ CH ₃ , hydroxyethyl formate-COOCH ₂ CH ₂ OH), and the like. Specific structural forms of the -C(COO-)-C- unit structure include -CH(COOH)-CH ₂ -, -CH(COONa)-CH ₂ -, -MeC(COOH)-CH ₂ -, - MeC(COONa)-CH ₂ -, -CH(COOCH ₃ )-CH ₂ -, -CH(COOCH ₂ CH ₂ OH)-CH ₂ -, -MeC(COOCH ₃ )-CH ₂ -, -MeC(COOCH ₂ CH ₂ OH)—CH ₂ —, etc., or any combination thereof. Here, Me is a methyl group. The linear main chain of one polymer molecule may contain only one type of unit structure (corresponding to a homopolymer), or may contain two or more types of unit structures. (corresponding to copolymer).

Acrylic monomer molecule: A monomer molecule used for synthesizing the above acrylic polymer, having a basic structure of C(COO-)=C, such as CH(COOH)=CH ₂ and CH(COONa)=CH ₂ , _CH3C (COOH)= _{CH2 ,} CH3C(COONa)= _CH2 , CH _{( COOCH3} ) _{= CH2} , CH( _COOCH2CH2OH )= _CH2 , CH3C( _COOCH3 )= CH ₂ , CH ₃ C(COOCH ₂ CH ₂ OH)=CH ₂ and the like.

Branched Chain: In the context of the present invention, a chain connected to a branch point and having independent ends. Branched chain and side branched chain in the present invention have the same meaning and can be used interchangeably. In the present invention, a branched chain means a side chain or a side group bonded to a linear main chain of a polymer, and the length and size of the branched chain are not particularly limited. It may be a short branched chain or a long branched chain with a large number of atoms. The structure of the branched chain is not particularly limited, and may be linear or have a branched structure. The branched chain may contain other side chains or side groups. Structural characteristics such as the number, length, size and degree of rebranching of the branched chains are preferably adjusted so as not to form a network structure as much as possible and increase the retention rate without causing accumulation of the branched chains, In this case, the flexible swing of the linear main chain can be exhibited smoothly.

Branched backbone: A branched backbone is one in which backbone atoms are sequentially linked in a covalent or non-covalent manner, from the branched chain ends to the backbone of the polymer. Branched backbones link functional groups at the ends of the polymer to the main chain of the polymer. The intersection of the branched backbone and the main chain is the branch point from which the branch is drawn. For example, the branched backbone between the purification medium and the polymer linear backbone is the affinity protein in the purification medium. —CH ₂ CH ₂ CH ₂ —NH—) and carbonyl groups (residues obtained by amidation reaction of carboxyl groups) can be sequentially linked to the polyolefin main chain of the polymer.

Branched chain ends include all branched chain ends. For the linear main chain, the end fixed to the main body of the magnetic microsphere and the other end of the linear main chain are always connected to one branch point, so the scope of the "branched chain end" of the present invention widely included in Accordingly, the polymer attached to the outer surface of the magnetic microsphere body of the present invention has at least one branch point.

Functional groups of polymer branches: have reactive activity, or have reactive activity after being activated, and can directly generate a covalent reaction with reactive groups of other raw materials, or have been activated It means that a covalent bond reaction with a reactive group of another raw material occurs later, and further a covalent bond is generated to connect. A functional group on the polymer branch is a specific binding site in one preferred embodiment.

The direct ligation system is a ligation system in which direct interaction occurs without intervening a spacer atom. The mode of interaction includes, but is not limited to, covalent mode, non-covalent mode, or combinations thereof.

An indirect connection scheme is a connection scheme formed via at least one linking element, in this case involving at least one spacer atom. Said linking elements include, but are not limited to, linking peptides, affinity complex links, and the like.

"Anchor" mode, such as anchoring, anchoring to, being anchored to, being anchored to, refers to a covalent mode of attachment.

The "ligation" / "binding" method such as with, is ligated, ligates to, ligates to, binds, captures, captures, etc. is not particularly limited, and includes covalent binding methods, non-covalent binding methods including but not limited to methods.

Covalent bonding method: A method of directly bonding with a covalent bond. The covalent bonding method includes, but is not limited to, dynamic covalent bonding method, and the dynamic covalent bonding method is a direct bonding method of dynamic covalent bonding.

Covalent bond: In addition to general covalent bonds such as amide bonds and ester bonds, reversible dynamic covalent bonds are also included. The covalent bond includes dynamic covalent bond. Dynamic covalent bonds are chemical bonds that are reversible and include, but are not limited to, imide bonds, hydrazone bonds, disulfide bonds, or combinations thereof. A chemical engineer can understand its meaning.

Non-covalent bonding methods: including but not limited to supramolecular interactions such as coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions.

Supramolecular interactions: including, but not limited to, coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof.

A linking element, also called a linking group, means an element for linking two or more non-adjacent groups and includes at least one atom. The linking method between the linking element and the adjacent group is not particularly limited, and includes, but is not limited to, covalent bonding methods, non-covalent bonding methods, and the like. The internal connection method of the connecting member is not particularly limited, and includes, but is not limited to, methods such as covalent bonding method and non-covalent bonding method.

Covalent linking element: All spacer atoms from one end of the linking element to the other are linked in a covalent manner.

Specific binding site: In the present invention, the specific binding site is a group or structural site having a binding function on the polymer branch chain, and the group or structural site specifically recognizes a specific target substance, Having a binding function, the specific binding can be achieved by bonding actions such as coordination, complexation, electrostatic forces, van der Waals forces, hydrogen bonding, covalent bonding, or other interactions.

Covalent Complex: Compounds obtained by direct or indirect covalent linkage, also called covalent conjugates.

Avidin-Purification Medium Covalent Complex: A compound linked in a covalent fashion, with avidin on one end and purification medium on the other, both linked directly via a covalent bond or via a covalent linking element. indirectly linked.

Avidin-affinity protein covalent complex E: an avidin-purification medium covalent complex with an affinity protein as a purification medium, or referred to as avidin-affinity protein complex E, with compounds linked in a covalent manner. with avidin at one end and an affinity protein at the other end, both of which are directly linked by a covalent bond or indirectly linked via a covalent linking group. The covalent linkage schemes include, but are not limited to, covalent bonds, connecting peptides, and the like. For example, streptavidin-protein A complex, streptavidin-protein A fusion protein, streptavidin-enhanced green fluorescent protein-protein A fusion protein (Protein A-eGFP-Streptavidin), Protein A-eGFP-Tamvavidin2, Protein G-eGFP-avidin fusion protein, Protein G-eGFP-Tamvavidin2, and the like.

Affinity complex: A non-covalent complex formed by two or more molecules with very strong affinity due to specific binding action, e.g. biotin (or biotin analogue) and avidin (or avidin (analogues). Methods for binding affinity complexes between biotin and avidin are well known to those skilled in the art.

A purified substrate, also called a target substance, is one that is separated from a mixed system. The purification substrate in the present invention is not particularly limited, but the purification substrate is preferably a proteinaceous substance (in this case, also referred to as a target protein).

The purification medium is capable of specifically binding the purification substrate, thereby capturing the purification substrate and further separating the purification substrate from the mixed system. Purification media attached to the branched ends of the polymers of the present invention are functional elements that have the ability to bind purification substrates. When the purification medium is covalently linked to adjacent groups, it is referred to as a functional group capable of binding the purification substrate.

Affinity protein: A protein that specifically binds to a target protein and has a relatively high affinity binding force. Examples include protein A, protein G, protein L, denatured protein A, denatured protein G, and denatured protein L. be done.

Protein A: Protein A, a 42 kDa surface protein, first found in the cell wall of Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topological structure, cell osmotic pressure and a binary system called ArlS-ArlR. It is used in biochemical research because of its ability to bind immunoglobulins. It can specifically bind to antibody Fc, is mainly used for antibody purification, and can be selected from any commercially available products. The so-called "Protein A", "SPA" and "Protein A" in the present invention are interchangeable.

Protein G: Protein G, an immunoglobulin binding protein, expressed in group C and group G streptococci, similar to protein A but with different binding specificities. It is a cell surface protein of 65 kDa (G148 protein G) and 58 kDa (C40 protein G) and is mainly used for antibody purification by specifically binding antibodies or certain functional proteins, selected from any commercial be able to.

Protein L: Protein L, limited to those that specifically bind to antibodies containing kappa (κ) light chains. In humans and mice, most antibody molecules contain a kappa light chain and a residual lambda light chain. It is mainly used for antibody purification and can be selected from any commercially available products.

Biotin: Biotin, capable of binding to avidin, has a strong binding force, and is excellent in specificity.

Avidin: avidin, which can bind to biotin, has strong binding force, and has excellent specificity, such as streptavidin (abbreviated as Streptavidin, SA), its analogues (for example, Tamvavidin2, abbreviated as Tam2), its denatured products, variants thereof, and the like.

A biotin analogue is a non-biotin molecule capable of forming a specific bond with avidin similar to "avidin-biotin", preferably a polypeptide or protein. ] series (eg, Strep[R]II, Twin-Strep-tag[R], etc.), and similar WN HPQFEK sequence-containing polypeptides. WNHPQFEK can be considered a variant sequence of WS HPQFEK.

An avidin analogue is a non-avidin molecule, preferably a polypeptide or protein, capable of forming a specific binding with avidin similar to "avidin-biotin". The avidin analogues include, but are not limited to, avidin derivatives, avidin homologues, avidin variants, and the like. Said avidin analogues are, for example, Tamavidin1, Tamavidin2, etc. (see literature: FEBS Journal, 2009, 276, 1383-1397).

Biotin-type tag: The biotin-type tag includes units of biotin, biotin analogues capable of binding to avidin, biotin analogues capable of binding to avidin analogues, and combinations thereof. A biotin-type tag can specifically bind avidin, an avidin analog, or a combination thereof. Therefore, a protein-based substance labeled with an avidin-type tag can be separated and purified, but is not limited to this.

Avidin-type tag: The avidin-type tag includes units of avidin, avidin analogues capable of binding to biotin, avidin analogues capable of binding to biotin analogues, and combinations thereof. Avidin-type tags can specifically bind biotin, biotin analogs, or combinations thereof. Therefore, a protein-based substance labeled with a biotin-type tag can be separated and purified, but it is not limited to this.

Polypeptide-type tags: A polypeptide-type tag of the present invention is a tag comprising a polypeptide tag or a derivative of a polypeptide tag. The polypeptide-type tag is a tag with a polypeptide structure composed of amino acids, and the amino acids may be natural amino acids or non-natural amino acids.

Protein-based tag: A protein-based tag of the invention is a tag comprising a protein tag or a derivative of a protein tag. The protein-type tag is a protein structure tag composed of amino acids, and the amino acids may be natural amino acids or non-natural amino acids.

Antibody-type tag: The antibody-type tag of the present invention is a tag containing an antibody-based substance, and can specifically bind to a target substance such as an antigen. Examples of the antibody-type tags further include antiEGFP nano-antibodies that can specifically bind to eGFP proteins.

Antigen-type tag: The antigen-type tag of the present invention is a tag containing an antigen-based substance and capable of specifically binding to an antibody-based substance.

Peptides are compounds in which two or more amino acids are linked by peptide bonds. In the present invention, peptide and peptide segment have equivalent meaning and can be used interchangeably.

Polypeptides are peptides composed of 10-50 amino acids.

Proteins are peptides composed of 50 or more amino acids. Fusion proteins are also proteins.

Derivatives of Polypeptides, Derivatives of Proteins: It is to be understood that any polypeptide or protein according to the invention also includes derivatives thereof, unless otherwise specified (eg, specifying a specific sequence). The polypeptide derivatives and protein derivatives include at least C-terminal containing tags, N-terminal containing tags, C-terminal and N-terminal containing tags. Here, the C-end refers to the COOH-end and the N-end refers to the _NH2 -end, and those skilled in the art will understand the meaning. Said tag may be a polypeptide tag or a protein tag. Some examples of tags include histidine tags (generally containing at least 5 histidine residues, such as 6xHis, HHHHHH, and also, for example, 8xHis tags), Glu-Glu, c-myc epitopes. (EQKLISEEDL), FLAG tag (DYKDDDDK), Protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 epitope label (V5 epitope, GKPIPNPLLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK) (hemagglutinin, YPYDVPDYA), β-galactosidase, thioredoxin, histidine site thioredoxin (His-patch thioredoxin), IgG binding domain (IgG-binding domain), intein-chitin binding domain (intein-chitin binding domain), T7 gene 10, glutathione-S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), etc. not.

A proteinaceous substance in the present invention is broadly defined as a substance containing a polypeptide or a protein fragment. For example, polypeptide derivatives, protein derivatives, glycoproteins, etc. are also included within the scope of proteinaceous substances.

Antibodies, Antigens: Antibodies and antigens of the present invention should be understood to include domains, subunits, fragments, single chains, single chain fragments and variants thereof, unless otherwise specified. For example, reference to an "antibody" includes fragments thereof, heavy chains, heavy chains lacking light chains (eg, nanobodies), complementarity determining regions (CDRs), etc., unless otherwise specified. For example, "antigen" further includes epitopes and epitope peptides, unless otherwise specified.

In the present invention, antibody-based substances refer to antibodies, antibody fragments, antibody single chains, single chain fragments, antibody fusion proteins, antibody fragments, as long as they can generate an antibody-antigen specific binding action. including, but not limited to, fusion proteins and the like, and derivatives and variants thereof.

In the present invention, antigen-based substances include, but are not limited to, antigens known to those skilled in the art, and substances that exert antigenic functions and specifically bind to antibody-based substances.

Anti-protein antibody: refers to an antibody that can specifically bind to a protein.

Anti-fluorescent protein nanobody: Refers to a nanobody that can specifically bind to a fluorescent protein.

Homology, unless otherwise stated, refers to having at least 50% homology, preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology. homologous, more preferably at least 80% homologous, more preferably at least 85% homologous, more preferably at least 90% homologous, and also for example at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. References include, for example, homologous sequences of the Ω sequences referred to in the description of the present invention. Homology here means sequence similarity, and may be numerically equivalent to identity.

A homologue refers to a substance with a homologous sequence, also called a homologue.

A "variant", variant, refers to a substance that has a different structure (including but not limited to making minor mutations) but retains or can essentially retain its original function or performance. Said variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. The manner in which related variants are obtained includes, but is not limited to, recombinations, deletions or deletions, insertions, shifts, substitutions, etc. of structural units. Said variants include, but are not limited to, modified products, genetically modified products, fusion products, and the like. Methods of genetic modification to obtain genetically modified products include, but are not limited to, genetic recombination (corresponding to genetically modified products), gene deletions or deletions, insertions, shifts, base substitutions, and the like. Gene mutation products, also called genetic variants, belong to one type of genetic modification products. One preferred form of said variant is a homologue.

Modified Products: Including but not limited to chemical modification products, amino acid modifications, polypeptide modifications, protein modifications and the like. The chemically modified products are products modified using chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry. Modification methods include ionization, salification, desalination, complexation, decomplexation, chelation, dechelation, addition reaction, substitution reaction, removal reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification. Specific examples include modification methods such as oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfurization.

Unless otherwise specified in the present invention, a "mutant" refers to a mutation product that retains or can essentially retain its original function or performance, and the number of mutation sites is not particularly limited. Said variants include, but are not limited to, genetic variants, polypeptide variants, protein variants. A mutant is a type of variant. The manner in which related variants are obtained includes, but is not limited to, recombination, deletion or deletion, insertion, shift, substitution, etc. of structural units. The structural unit of genes is the base, and that of polypeptides and proteins is the amino acid. Types of genetic mutations include, but are not limited to, gene deletions or deletions, insertions, shift coding, base substitutions, and the like.

"Modified" products include, but are not limited to, derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, which can retain their original function or performance, and which Functionality or performance may be optimized or changed.

Eluate (eg target protein): to elute the target protein, after elution the target protein is present in the eluate.

Wash solution (eg target protein): to elute the impurity protein, which after elution is carried away by the wash solution.

Avidity: Binding capacity, eg, binding capacity of a magnetic microsphere to a protein.

Affinity potency: Substrate concentration when magnetic microspheres bind only 50% of the substrate using substrate solutions with different concentration gradients.

IVTT: In vitro transcription and translation, an in vitro transcription and translation system, ie a cell-free protein synthesis system. A cell-free protein synthesis system uses an exogenous target mRNA or DNA as a template for protein synthesis, and adds substances such as substrates and transcription- and translation-related protein factors required for protein synthesis through artificial control to achieve target protein synthesis. can be done. The cell-free protein synthesis system of the present invention is not particularly limited, and any cell-free protein synthesis system based on yeast cell extract, E. coli cell extract, mammalian cell extract, plant cell extract, insect cell extract, or Any combination may be used.

In the present invention, "translation-related enzymes", translation-related enzymes (TRENs), means enzymatic substances necessary for the process of synthesizing a protein product from a nucleic acid template, and is not limited to enzymes necessary for the translation process. do not have. Nucleic acid template: Also called genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates and combinations thereof.

Flow-through: Clear fluid collected after incubating a system containing magnetic beads and target proteins, of which it contains residual target proteins not captured by the magnetic beads. For example, in Example 2, a solution containing a complex of an avidin-affinity protein is added to an affinity column, and is the solution that exits after passing through the column. The 1st, 2nd and 3rd run out solutions are shown.

RFU is Relative Fluorescence Units.

eGFP: enhanced green fluorescence protein. In the present invention, the eGFP broadly includes wild type and variants thereof, including but not limited to wild type and variants thereof.

mEGFP: A206K variant of eGFP.

"Optionally" indicates that the technical solution of the present invention can be implemented as a selection criterion, and may or may not be present. In the present invention, "arbitrary method" indicates that the present invention can be implemented by applying the technical idea of the present invention.

In the present invention, preferred embodiments such as "preferred (for example, prefer, preferred, preferred, preferred, etc.)", "preferably", "more preferred", "further preferred", and "most preferred" refer to the scope of the invention and It does not limit the scope of protection in any way, and does not limit the scope or embodiments of the present invention, but merely exemplifies some embodiments.

In the description of the present invention, "preferred one", "preferred form", "preferred embodiment", "preferred example", "preferred example", "in a preferred mode of implementation", "in some preferred examples ", "in some preferred forms", "preferred", "preferred", "preferred", "more preferred", "more preferred", "further preferred", "most preferred", etc. and schematic recitations such as "one embodiment", "one form", "illustration", "example", "example", "example", "for example", "example", "such as" The scheme likewise does not in any way constitute a limitation on the scope and protection of the invention, and the specific features described in each form are included in at least one specific embodiment of the invention. In the present invention, the specific features described in each form can be combined in any suitable form in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred form can be combined in any suitable form.

In the present invention, "any combination thereof" means numerically "greater than 1", range-wise "any one may be selected, or a group consisting of at least two You may."

In the present invention, the description of "one or more" such as "one or more", "one or more" means "at least one", "at least one", "a combination thereof", "or a combination thereof", " and any combination thereof", "or any combination thereof", "and any combination thereof", etc., have the same meaning and can be used interchangeably, and that the quantity is equal to "one" or "one or more" indicates

In the present invention, "or/and" is employed, and "and/or" represents "arbitrarily one or a combination thereof" and indicates at least one of them.

The conventional technical means described in the manner of "usually", "usually", "general", "always", "usually", etc. described in the present invention are all cited as reference for the content of the present invention, Unless otherwise stated, it can be regarded as a preferred form of some technical features of the present invention, and it should be noted that it does not constitute any limitation on the scope and protection scope of the invention in any way.

All documents mentioned in the present invention and documents directly or indirectly cited from those documents are incorporated by reference in this application, and each document appears to be individually cited by reference.

It should be understood that within the scope of the present invention, each of the above technical features of the present invention and each of the technical features specifically described below (including but not limited to the examples) can only be combined with each other to form new or preferred technical solutions and implement the present invention. Due to space limitations, I will not repeat all of them.

1. A first aspect of the present invention provides biomagnetic microspheres, said biomagnetic microspheres comprising a magnetic microsphere body, the outer surface of said magnetic microsphere body having a linear backbone and branched chains. one end of the linear main chain is fixed to the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the polymer of the magnetic microsphere is ligated to the branched end of biotin or a biotin analogue.

The biomagnetic microspheres of the first aspect of the invention are also called biotin magnetic microspheres or biotin magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG.

Said biotin or biotin analogue can not only serve as a purification medium, but can also be linked as a linking element to still other types of purification media.

Most commercial microspheres employ agarose-based materials, as compared to gel-based porous materials commonly used today, such as agarose-based materials. Porous materials have a rich pore structure, which provides a large specific surface area and high binding capacity for purification matrices, but correspondingly, protein molecules are porous when adsorbing or eluting proteins. Requires additional entry or exit through complex pore passages inside the material, which is more time consuming and prone to retention. In contrast, the binding site for target protein capture provided by the present invention utilizes only the outer surface space of the biomagnetic microspheres, and does not need to pass through complicated network passages during adsorption and elution, and the elution solution can be released directly into the water, thereby greatly reducing the elution time, improving the elution efficiency, reducing the retention rate and improving the purification yield.

1.1. Magnetic Microsphere Bodies In the present invention, the volume of the magnetic microsphere bodies can be of any viable particle size.

A relatively small particle size helps ensure that the magnetic microspheres are suspended in a mixed system and have more intimate contact with the protein product, improving the capture efficiency and binding rate of the protein product. In some preferred embodiments, the diameter size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0 .5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm , 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm , 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 500 μm, 500 μm , 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm, any one particle size scale or any two particle size scales (with deviations of ±25%, ±20%, ±15% , may be ±10%). Unless otherwise stated, the diameter size refers to the average size.

The volume of the magnetic microsphere body can be of any viable particle size.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.1-10 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.2-6 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.4-5 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.5-3 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.2-1 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.5-1 μm.

In some preferred forms, said magnetic microsphere bodies have an average diameter of and the approximate number may be ±25%, ±20%, ±15%, ±10%.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 1 μm to 1 mm.

In some preferred forms, the diameter of the magnetic microsphere body is e.g. %.

Different magnetic materials can provide different types of activation sites, can produce different modes of binding with the purification medium, and have different abilities to disperse and sediment with a magnet, and can also vary with the purification substrate type. Selectivity can be generated.

Magnetic microsphere bodies, and magnetic microspheres comprising magnetic microsphere bodies, can be rapidly positioned, guided, and separated under the action of an applied magnetic field, while multiple magnetic microsphere surfaces are formed on the magnetic microsphere surfaces by methods such as surface modification or chemical polymerization. can be provided with active functional groups such as hydroxyl, carboxyl, aldehyde, and amino groups, and the magnetic microspheres can bind biologically active substances such as antibodies and DNA by covalent or non-covalent methods. be able to.

In some preferred forms, the magnetic microsphere body is a magnetic material coated with _SiO2 . Here, the _SiO2 coating layer may contain a silane coupling agent having an active site on itself.

In some preferred forms, the magnetic material is an iron compound (e.g., iron oxide), iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide, manganese alloy, zinc oxide, gadolinium oxide. , chromium oxide, and combinations thereof.

In some preferred forms, the iron oxide is, for example, magnetite (Fe ₃ O ₄ ), maghemite (γ-Fe ₂ O ₃ ) or a combination of the two oxides, preferably triiron tetroxide. be.

In some preferred forms, the magnetic material is Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , AlNi(Co), FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo , MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), _FeSiAl , _BaO.6Fe2O3 , _SrO.6Fe2O3 , _{PbO.6Fe2O3 , GdO} and their Selected from a combination. Here, Re is rhenium, which is a rare earth element.

1.2. A polymeric structure that provides a large amount of branched chain ends The outer surface of the magnetic microsphere body has at least one polymer with a linear backbone and branched chains, and one end of the linear backbone is the magnetic microsphere body The other end of the polymer is free from the outer surface of the magnetic microsphere body.

The term "fixed to" refers to being "fixed to" the outer surface of the magnetic microsphere body in a covalent manner.

In some preferred forms, the polymer is covalently attached to the outer surface of the magnetic microsphere body either directly or indirectly via a linking element.

Said polymer has a linear backbone, in which case the polymer not only has the high flexibility of the linear backbone, but also has the advantage of high-fold amplification of the number of branched chains, enabling high-rate and high-throughput binding. , high-efficiency, high-ratio (high-yield) separations can be better achieved.

For the magnetic microspheres of the present invention, one end of the polymer is covalently attached to the outer surface of the magnetic microsphere body, and the remaining ends containing all branched chains and all functional groups are both dissolved in a solution, and the magnetic microsphere body is The molecular chains can be sufficiently stretched and oscillated, and the molecular chains can be sufficiently contacted with other molecules in the solution, further enhancing the capture of the target protein. . When the target protein is eluted from the magnetic microspheres, it directly escapes from the confinement of the magnetic microspheres and enters directly into the eluate. Polymers covalently anchored at one end by such linear backbones (some In preferred embodiments, a single single polymer linear backbone is covalently anchored, and in some other preferred embodiments, two or three linear backbones are covalently drawn from the anchored ends of the backbone. ) can effectively reduce the stacking of molecular chains, enhance the extension and rocking of molecular chains in solution, enhance the capture of target proteins, and reduce the retention rate and retention time of target proteins during elution. do.

1.2.1. Polymer Backbone of Biomagnetic Microspheres Provided by the Present Invention In some preferred forms, the linear backbone is a polyolefin backbone or an acrylic polymer backbone.

In some other preferred forms, the linear backbone of the polymer is an acrylic polymer backbone. It may be a polyolefin backbone (the linear backbone is only carbon atoms) or it may contain heteroatoms in the linear backbone (the heteroatoms are non-carbon atoms).

In some preferred forms, the polymer backbone is a polyolefin backbone. The monomer unit of the acrylic polymer is an acrylic monomer molecule such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof. The acrylic polymer may be obtained by polymerizing any of the above monomers, or may be obtained by copolymerizing an appropriate combination of the above monomers.

In some preferred forms, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin main chain is a main chain provided to a polymer of any one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, and methacrylate monomer. , or the backbone provided to the polymerization product of a combination thereof (the backbone provided to the copolymer thereof), or the backbone of the copolymer formed when the above monomers participate in the polymerization . Polymerization products obtained by combining the above monomers include, for example, acrylic acid-acrylic acid ester copolymer, methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate. A copolymer etc. are mentioned. Examples of copolymers formed through polymerization of the above monomers include maleic anhydride-acrylic acid copolymers.

In some preferred forms, the linear backbone is a polyolefin backbone and is provided from an acrylic polymer backbone.

In some preferred forms, the linear backbone is an acrylic polymer backbone.

In some other preferred forms, the polymer backbone is an acrylic polymer backbone. It may be a polyolefin backbone (the backbone is only carbon atoms) and may contain heteroatoms (heteroatoms: non-carbon atoms) in the backbone.

In some other preferred forms, the polymer backbone is a block copolymer backbone comprising polyolefin blocks, such as polyethylene glycol-b-polyacrylic acid copolymer (which belongs to the range of acrylic copolymers). ). It is preferred that the flexible rocking of the linear backbone is smoothly exerted without causing branch chain build-up and increasing the residence time or/and rate.

In some other preferred forms, the polymer backbone is a polycondensed backbone. The polycondensation type main chain means a linear main chain that can be formed by a polycondensation reaction between monomer molecules or oligomers. The polycondensation-type main chain may be homotype or copolymer type. For example, polypeptide chains, polyamino acid chains, and the like. Specific examples include ε-polylysine chains, α-polylysine chains, γ-polyglutamic acid chains, polyaspartic acid chains, and aspartic acid/glutamic acid copolymers.

The number of linear backbones to which one binding site on the outer surface of the magnetic microsphere body can be covalently bound may be one or more.

In some preferred forms, one binding site on the outer surface of the magnetic microsphere body can pull out only one linear backbone, providing a large working space for the linear backbone.

In some other preferred forms, one binding site on the outer surface of the magnetic microsphere body pulls out only two linear backbones, providing as much activity space for the linear backbones as possible.

The backbone of the polymer is covalently attached at one end to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), and the remaining ends containing all branches and all functional groups are both dissolved in solution and magnetically Distributed in the outer space of the beads, the molecular chains can be sufficiently stretched and oscillated, which allows the molecular chains to make sufficient contact with other molecules in the solution, further enhancing the capture to the target protein. be able to. When the target protein is eluted from the magnetic beads, it is directly released from the confinement of the magnetic beads and directly into the eluate. Polymers covalently anchored by one end of the linear backbones provided herein (most preferably single A single polymer linear backbone is covalently anchored, and preferably two or three linear backbones are covalently drawn to the anchored end of the backbone) effect stacking of the molecular chains. can be reduced exponentially, enhancing the stretching and rocking of the molecular chains in solution, enhancing capture of the target protein, and reducing the retention rate and residence time of the target protein during elution.

1.2.2. Polymer branched chains of the biomagnetic microspheres provided by the present invention The number of said branched chains is the size of the magnetic microsphere body, the backbone structure type of the polymer, the chain density on the outer surface of the magnetic microsphere body of the polymer (particularly the branched chains). density).

The number of polymer branches is multiple and at least three. The number of side branches is related to the size of the magnetic microsphere, the length of the polymer backbone, the linear density of the side branches along the polymer backbone, and the polymer to factors such as chain density at the outer surface of the magnetic microsphere. Related. The number of polymer branched chains can be controlled by controlling the charging ratio of raw materials.

The branched polymer has at least three branches.

Each branched chain end may or may not be independently bound to a purification medium.

When a purification medium is bound to the branched chain ends, each branched chain end may be independently bound directly to the purification medium, or may be indirectly bound via a linking element. good.

When purification media are attached to the ends of branched chains, the number of purification media may be one or more.

In some preferred forms, one molecule of said branched polymer is associated with at least three purification media.

1.3. Binding Mode of Biotin or Biotin Analog The mode in which the biotin or biotin analogue is linked to the branched end of the polymer is not particularly limited.

The manner in which the biotin or biotin analog is linked to the branched ends of the polymer includes, but is not limited to, covalent bonding, supramolecular interactions, or combinations thereof.

In some preferred forms, said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof. be.

In some preferred forms, the branched chains of the polymer are covalently attached to biotin or biotin analogues by functional group-based covalent bonds, covalently attaching biotin or biotin analogues to the ends of the polymer branches. It can be obtained by covalent reaction of biotin or biotin analogues with functional groups contained in branched chains of polymer molecules on the outer surface of biomagnetic microspheres. Here, one of the preferred embodiments of said functional group is a specific binding site (see "Nouns and Terms" section of specific embodiments for definition).

The covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group. Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. One of the preferred salt forms of the carboxyl group is a sodium salt form, such as COONa, and the preferred salt form of the amino group may be an inorganic salt form or an organic salt form. may also include, but are not limited to, hydrochloride, hydrofluoride, and like forms. The "combination of functional groups" refers to all branches of all polymer molecules on the outer surface of one magnetic microsphere, which can participate in the formation of covalent bonds based on different functional groups. Taking biotin as an example, all biotin molecules on the outer surface of one biotin magnetic microsphere can be covalently bound with different functional groups, but one biotin molecule can only be bound with one functional group. .

2. A second aspect of the present invention is based on said biomagnetic microspheres provided in the first aspect of the present invention, wherein said biotin or biotin analogue is further linked to a purification medium as a linking element. provide a sphere. That is, the branched ends of the polymer are linked to the purification medium via a linking element, the linking element comprising biotin or a biotin analogue.

purification element
A purification medium is a functional requirement that specifically captures a target substance from a mixed system, ie, a specific binding can occur between the purification medium and target substance molecules to be separated and purified. The captured target substance molecules are eluted and released under appropriate conditions, thereby achieving the purpose of separation and purification.

When the target substance is a protein substance, the purification medium can form a specific binding action with the target protein itself or with a purification tag carried by the target protein. Therefore, any substance that can be used for a target protein purification tag can be a selectable format for a purification medium, and a peptide or protein used as a purification medium can be a selectable format for a purification tag for a target protein. can do.

2.1. Types of Purification Media The purification media may contain, but are not limited to, avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In some preferred forms, the purification medium comprises avidin, an avidin analogue capable of binding to biotin or an analogue thereof, biotin, a biotin analogue capable of binding to avidin or an analogue thereof, an affinity protein, an antibody, an antigen, a DNA , or a combination thereof.

In some preferred forms, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin. do.

In some preferred forms, the avidin is streptavidin, denatured streptavidin, streptavidin analogs, or a combination thereof.

Said avidin analogues are, for example, tamavidin 1, tamavidin 2 and the like. Ｔａｍａｖｉｄｉｎ１及びＴａｍａｖｉｄｉｎ２は、Ｙａｍａｍｏｔｏらが２００９年に発見したビオチン結合能力を有するタンパク質であり（Ｔａｋａｋｕｒａ Ｙ ｅｔ ａｌ．Ｔａｍａｖｉｄｉｎｓ：Ｎｏｖｅｌ ａｖｉｄｉｎ－ｌｉｋｅ ｂｉｏｔｉｎ－ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎｓ ｆｒｏｍ ｔｈｅ Ｔａｍｏｇｉｔａｋｅ ｍｕｓｈｒｏｏｍ［Ｊ］．ＦＥＢＳ Ｊｏｕｒｎａｌ，２００９， 276, 1383-1397), they have a strong affinity for biotin similar to streptavidin. The thermostability of Tamavidin2 is superior to that of streptavidin, and its amino acid sequence can be retrieved from relevant databases, such as UniProt B9A0T7, and can also be codon-transformed and optimized by an optimization program to obtain the DNA sequence.

The biotin analogues are, for example, the WSHPQFEK sequence or its variant sequences, the WRHPQFGG sequence or its variant sequences, and the like.

In some preferred forms, the purification medium is a polypeptide tag, protein tag, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein.

Examples of said affinity proteins include, but are not limited to, protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, and the like.

The definitions of the antibodies and antigens refer to the term part, domain, subunit, fragment, heavy chain, light chain, single chain fragment (e.g. nanobody, light chain deleted heavy chain, heavy chain variable region, complementarity determining regions, etc.), epitopes, epitope peptides, variants of any of the foregoing, and the like.

In some preferred embodiments, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a tag containing a WSHPQFEK sequence, a tag containing a variant sequence of WSHPQFEK. , a tag comprising a WRHPQFGG sequence, a tag comprising a variant sequence of WRHPQFGG, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or combinations thereof or variants thereof.

In some preferred forms, said protein tag is any tag or variant thereof selected from an affinity protein, a SUMO tag, a GST tag, an MBP tag, or combinations thereof, more preferably said affinity protein is , protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

In some preferred embodiments, the antibody-based tag is an antibody, a fragment of an antibody, a single chain of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any, or A variant of either.

In some preferred forms, the antibody-based tag is an anti-protein antibody.
In some preferred forms, the antibody-based tag is an anti-fluorescent protein antibody.
In some preferred forms, the antibody-based tag is a nanobody.
In some preferred forms, the antibody-based tag is an anti-protein nanobody.
In some preferred forms, the antibody-based tag is an anti-fluorescent protein nanobody.
In some preferred forms, the antibody-based tag is an anti-green fluorescent protein or variant thereof antibody.
In some preferred forms, said antibody-based tag is an Fc fragment.

2.2. Supporting Method of Purification Medium The method in which the purification medium is linked to the biotin or biotin analogue is not particularly limited.

The manner in which the purification medium is linked to the biotin or biotin analog includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or combinations thereof.

In some preferred forms, said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof. be.

In some preferred forms of the biomagnetic microspheres, the purification media are linked to the branched ends of the polymer by linking elements comprising affinity complexes.

In some preferred forms, the biotin or biotin analogue is bound to avidin or an avidin analogue by affinity complexing, and the purification medium directly or indirectly binds to the avidin or avidin analogue.

In some preferred forms, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction .

In some preferred forms, the selection criteria for the affinity conjugate are to provide a highly specific, high affinity, yet chemically conjugable site whereby the affinity conjugate is covalently attached to the polymer branch ends. or can be covalently attached to the outer surface of the magnetic microsphere body after being chemically modified, e.g. be. For example, combinations of substances from biotin or its analogues and avidin or its analogues, antigens and antibodies and the like.

If the loading scheme involves dynamic covalent bonding, supramolecular interactions (especially affinity complex interactions), it forms a reversible loading scheme, and purification media can be unloaded from branched chain ends under certain conditions. , further update or replace.

Renewal of purification media corresponds to regeneration of magnetic microspheres, and the type of purification media before and after renewal is the same.

The exchange of purification media corresponds to changing the magnetic microspheres, and the types of purification media before and after the exchange are different.

In some preferred forms, the purification medium is avidin and further comprises biotin that binds to the avidin, wherein the biotin is a linking member and the binding of the affinity complex between biotin and avidin.

In some preferred forms, the purification medium is an affinity protein and further comprises avidin linked to the affinity protein and biotin linked to the avidin, wherein binding of the affinity complex between biotin and avidin form an action, with the affinity complex as the linking element.

In some preferred forms, biotin, avidin, and purification media are sequentially linked to the branched ends of the polymer of the biomagnetic microspheres. In a further preferred form, said purification medium is an antibody or antigen. Modes of linking the avidin with the purification medium include, but are not limited to, covalent bonds, non-covalent bonds, linking elements, or combinations thereof.

In some preferred forms, the purification medium comprises nucleic acids, oligonucleotides, peptide nucleic acids, nucleic acid aptamers, deoxyribonucleic acids, ribonucleic acids, leucine fasteners, helix turn helix motifs, zinc finger motifs, biotin, anti-biotin proteins, streptavidin, The biomagnetic microspheres are linked to the polymer branched ends of the biomagnetic microspheres by linking elements, including but not limited to combinations thereof, such as anti-hapten antibodies. Of course, said linking elements are selected from double-stranded nucleic acid structures, double helices, homohybrids or heterohybrids (DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA homohybrids or heterohybrids), or combinations thereof.

2.3. Mechanism of Action of the Purification Medium The force with which the purification medium captures target molecules in the reaction-purification mixture system is selected from, but not limited to, covalent bonds, supramolecular interactions, and combinations thereof.

In some preferred forms, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction .

In some preferred forms, the target substance has biotin-avidin binding capacity, Streg tag-avidin binding capacity, avidin-affinity protein binding capacity, histidine tag-metal ion affinity, antibody-antigen binding capacity, or combinations thereof. The biomagnetic microspheres are bound to the polymer branched chain ends by a force method. The Streg tag mainly includes, but is not limited to, a peptide tag that forms a specific binding activity with avidin or its analogue developed by IBA, and usually includes a WSHPQFEK sequence or a modified sequence thereof.

2.4. Regeneration and Reuse of Purification Media If the purification media is linked to the polymer branched ends of the biomagnetic microspheres of the invention by reversible methods such as affinity complexes, dynamic covalent bonding, etc., under appropriate conditions, the purification media can be combined with the polymer. The branched ends can be eluted and recombined with new purification media.

Affinity complex interactions are exemplified by affinity complex interactions between biotin and streptavidin.

The extremely strong affinity between biotin and streptavidin is the binding action of typical affinity complexes, stronger than common non-covalent binding actions and at the same time weaker than covalent binding actions, thereby allowing purification media to escape from magnetic beads. Not only can it bind tightly to the polymer branch ends on the surface, but also achieve synchronous release of the purification medium by eluting streptavidin from the specific binding site of biotin when the purification medium needs to be replaced. , further liberating the activation site of new avidin-purification media covalent complexes (e.g. streptavidin-tagged purification media), thereby enabling rapid recovery of magnetic bead purification performance and target substances (e.g. antibodies) significantly reduce the cost of separation and purification of The process of eluting the purification medium-modified biomagnetic microspheres and removing the avidin-purification medium covalent complexes, thereby recapturing the biotin or biotin analogue-modified biomagnetic microspheres is characterized by biotin magnetic properties. called microsphere regeneration. The renatured biotin-magnetic microspheres have released biotin active sites and can rebind avidin-purification medium covalent complexes, yielding again purification medium-modified biomagnetic microspheres ( (corresponding to regeneration of biomagnetic microspheres), fresh purification media can be provided, providing new target substance binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated and used, ie, the purification media can be exchanged and reused.

2.4. Purification substrate (preferably proteinaceous material)
The purification substrate of the present invention is a substance that is captured and separated by the magnetic microspheres of the present invention, and is particularly limited as long as it is a purification medium that can specifically bind to the magnetic microspheres of the present invention. isn't it.

When said purification substrate is a protein-based substance, the purification substrate is also called target protein.

2.4.1. Purification Tags in Target Proteins The target protein may not carry a purification tag, in which case the target protein itself should be captured in a purification medium in magnetic microspheres. For example, "target protein, purification medium" may be combinations such as "antibody, antigen", "antigen, antibody", "avidin or analogue thereof, biotin or analogue thereof".

In some preferred forms, the target protein is tagged with a purification tag, and the purification tag is capable of specifically binding to the purification medium. In one target protein molecule, the number of the purification tags is one, two or more, and when two or more purification tags are included, the types of purification tags are one, two or multiple. As long as the amino acid sequences of the tags are different, they are regarded as different types of tags.

Purification tags in the target protein include histidine tag, avidin, avidin analogs, Streg tag (tag containing WSHPQFEK sequence or variants thereof), tag containing WRHPQFGG sequence or variants thereof, tag comprising RKAAVSHW sequence or variants thereof, FLAG tag. Or its variants, C tag and its variants, Spot tag and its variants, GST tag and its variants, MBP tag and its variants, SUMO tag and its variants, CBP tag and its variants, HA tag and its variants, Avi tag and its The above groups of tags include, but are not limited to variants, affinity proteins, antibody-based tags, antigen-based tags, and combinations thereof. It can also be selected from the purification tags disclosed in US6103493B2, US10065996B2, US8735540B2, US20070275416A1, including but not limited to Streg tags and variants thereof.

The purification tag can be fused through the N-terminus or the C-terminus.

Said histidine tag generally comprises at least 5 histidine residues, such as a 5xHis tag, a 6xHis tag, an 8xHis tag, and the like.

The octapeptide WRHPQFGG can specifically bind core streptavidin.

The Streg tag is capable of forming a specific binding action with avidin or an analogue thereof, including WSHPQFEK or variants thereof in said Streg tag. For example, WSHPQFEK-(XaaYaaWaaZaa) _n -WSHPQFEK, where Xaa, Yaa, Waa, Zaa are each independently any amino acid, XaaYaaWaaZaa comprises at least one amino acid, and (XaaYaaWaaZaa) _n is comprising at least 4 amino acids, where n is selected from 1 to 15 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) , (XaaYaaWaaZaa) _n include (G) ₈ , (G) ₁₂ , GAGA, (GAGA) ₂ , (GAGA) ₃ , (GGGS) ₂ and (GGGS) ₃ . Ｓｔｒｅｇタグとしては、例えば、ＷＳＨＰＱＦＥＫ、ＷＳＨＰＱＦＥＫ－（ＧＧＧＳ） _ｎ －ＷＳＨＰＱＦＥＫ、ＷＳＨＰＱＦＥＫ－ＧＧＧＳＧＧＧＳＧＧＳＡ－ＷＳＨＰＱＦＥＫ、ＳＡ－ＷＳＨＰＱＦＥＫ－（ＧＧＧＳ） _２ ＧＧＳＡ－ＷＳＨＰＱＦＥＫ、ＷＳＨＰＱＦＥＫ－ＧＳＧＧＧ－ＷＳＨＰＱＦＥＫ－ＧＬ－ＷＳＨＰＱＦＥＫ、ＧＧＳＡ－ＷＮＨＰＱＦＥＫ－ GGGSGSGGSA-WSHPQFEK-GS, GGGS-WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK, and the like.

The sequence of the FLAG tag is DYKDDDDK. Variant sequences of the FLAG tag include, for example, DYKDHD-G-DYKDHD-I-DYKDDDDK.

The sequence of the Spot tag is PDRVRAVSHWSS.

The C-tag contains an EPEA sequence.

The GST tag means a glutathione S-transferase tag.

The MBP tag is a maltose binding protein tag.

The SUMO tag is a known small ubiquitin-like modifier and one of the key members of the ubiquitin family of polypeptide chain superfamily. In primary structure, SUMO and ubiquitin share 18% homology, but their tertiary structures and their biological functions are quite similar.

The sequence of the CBP tag is KRRWKKNFIAVSAANRFKKISSSGAL.

The HA tag sequence is YPYDVPDYA.

The Avi-tag is a small tag composed of 15 known amino acid residues, which is specifically recognized by the biotin ligase BirA.

Antibody tags include complete antibody structures (whole antibodies), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g. nanobodies, heavy chains with light chains deleted, heavy chain variable regions, complementarity determining regions, etc.).

Antigenic tags include, but are not limited to, complete antigen structures (complete antigens), domains, subunits, fragments, heavy chains, light chains, single chain fragments (eg, epitopes, etc.), and the like.

In some preferred forms, one purification tag is attached to the N-terminus or C-terminus of the target protein, or both ends are attached to purification tags.

Any of the various purification tags described in this section can be candidates for purification media in the magnetic microspheres of the present invention.

2.4.2. Type of Target Protein The target protein may be a natural protein or modified product thereof, or an artificial synthetic sequence. The origin of the natural protein is not particularly limited, and includes but is not limited to eukaryotic cells, prokaryotic cells, and pathogens, among which eukaryotic cell origins include mammalian cells, plant cells, yeast cells, insect cells, and lineages. Said mammalian cells, including but not limited to worm cells, and combinations thereof, may be derived from murine sources (including rats, mice, guinea pigs, golden gophers, hamsters, etc.), rabbit sources, monkey sources, human sources, Including, but not limited to, swine sources, sheep sources, bovine sources, dog sources, horse sources, and the like. Said pathogens include viruses, chlamydia, mycoplasma, and the like. Said viruses include HPV, HBV, TMV, coronaviruses, rotaviruses and the like.

The types of target proteins include polypeptides (the term "target protein" in the present invention broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagen, polypeptides. Including, but not limited to, amino acids, vaccines, etc., partial domains of any of the above proteins, subunits or fragments of any of the above proteins, and variants of any of the above proteins. The "subunit or fragment of any of the above proteins" includes subunits or fragments of "a partial domain of any of the above proteins." The "variants of any of the above proteins" include variants of "a partial domain of any of the above proteins, subunits or fragments of any of the above proteins". The "variants of any of the above proteins" include, but are not limited to, variants of any of the above proteins. In the present invention, situations and meanings of two or more "above" in succession in other positions are similarly explained.

The structure of the target protein may be a complete structure, or may be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, and the like. Incomplete antibody structures include, for example, _nanobodies (heavy chain antibodies with light chains deleted, VHH, retaining the full antigen-binding ability of heavy chain antibodies), heavy chain variable regions, complementarity determination region (CDR) and the like.

For example, target proteins that can be synthesized by the in vitro protein synthesis system according to the present invention are selected from, but limited to, any of the following proteins, fusion proteins in any combination, and compositions in any combination: not. namely, luciferase (eg, firefly luciferase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), ammonia acyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase. , mouse catalase), actin, antibodies, antibody variable regions (e.g. antibody single chain variable regions, scFV), antibody single chains and fragments thereof (e.g. antibody heavy chains, nanobodies, antibody light chains) , alpha-amylase, enterobacter A, hepatitis C virus E2 glycoprotein, insulin and its precursors, glucagon-like peptide (GLP-1), interferons (including interferon alpha, interferon beta, interferon gamma, such as interferon alpha A) ), interleukins (such as interleukin-1β, interleukin-2, interleukin-12), lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, A partial domain of any of the foregoing proteins, such as xylanase, β-galactosidase, LacZ, e.g. E. coli β-galactosidase, a subunit or fragment of any of the foregoing proteins, or a variant of any of the foregoing (defined above) As such, said variants include variants, eg, luciferase variants, eGFP variants, which variants may also be homologues). Examples of the ammonia acyl-tRNA synthetase include human lysine-tRNA synthetase and human leucine-tRNA synthetase. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN109423496A. A composition of any of the above combination modalities may comprise any of the proteins described above and may comprise a fusion protein of any of the above combination modalities.

In some preferred embodiments, a target protein with fluorescent properties, such as one of GFP, eGFP, mScarlet, etc., or analogues or variants thereof, is used to assess the protein synthesis capacity of the in vitro protein synthesis system. .

The application fields of said target protein include biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, biotechnology, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanotechnology, cosmetics, food, Including but not limited to fields such as food additives, nutrients, agriculture, feed, daily necessities, laundry, environment, chemical dyeing and fluorescent labeling.

2.4.3. Mixed Systems Containing Target Proteins The magnetic microspheres of the invention can be used to separate target proteins from their mixed systems. The target protein is not limited to one substance, but may be a combination of multiple substances, provided that the purpose of purification is to obtain this composition, or the form of this composition can meet the purification requirements. may

The mixed system containing the target protein is not particularly limited, and the purification medium for the magnetic microspheres of the present invention only needs to be able to specifically bind to the target protein. It is also necessary that there is no specific binding or non-specific binding activity of the compound with other substances.

In embodiments of the present invention, the mixed system containing the target protein may be of natural origin, artificially constructed or derived mixed system.

For example, a specific protein can be separated and purified from commercially available serum.

For example, the target protein can be separated from the post-reaction system of an in vitro protein synthesis system.

A specific embodiment of said in vitro protein synthesis system further includes, but is not limited to, the E. coli-based cell-free protein synthesis system described, for example, in WO2016005982A1. Other citations of the present invention are in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marcianas cited in the direct and indirect citations. and are not limited to, all of which are incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, in the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45", part "2.1 Systems and Advantage 2 on page 7 to 8" Any of the in vitro protein synthesis systems can be an in vitro protein synthesis system to practice the present invention, including, but not limited to, the in vitro cell-free protein synthesis systems described in the literature.例えば、（本発明と矛盾しない限り、以下の文献及びその参考文献をその全体及びすべての目的のために引用する）文献ＣＮ１０６９７８３４９Ａ、ＣＮ１０８５３５４８９Ａ、ＣＮ１０８６９０１３９Ａ、ＣＮ１０８９４９８０１Ａ、ＣＮ１０８６４２０７６Ａ、ＣＮ１０９０２２４７８Ａ、ＣＮ１０９４２３４９６Ａ、ＣＮ１０９４２３４９７Ａ、ＣＮ１０９４２３５０９Ａ、ＣＮ１０９８３７２９３Ａ 、ＣＮ１０９９７１７８３Ａ、ＣＮ１０９９８８８０１Ａ、ＣＮ１０９９７１７７５Ａ、ＣＮ１１００９３２８４Ａ、ＣＮ１１０４０８６３５Ａ、ＣＮ１１０４０８６３６Ａ、ＣＮ１１０５５１７４５Ａ、ＣＮ１１０５５１７００Ａ、ＣＮ１１０５５１７８５Ａ、ＣＮ１１０８１９６４７Ａ、ＣＮ１１０８４５６２２、ＣＮ１１０９３８６４９Ａ、ＣＮ１１０９６４７３６Ａ、ＣＮ１１１３７８７０６Ａ、ＣＮ１１１３７８７０７Ａ、ＣＮ１１１３７８７０８Ａ、ＣＮ１１１７１８４１９Ａ、ＣＮ１１１７４８５６９Ａ、ＣＮ２０１９１０７２９８８１３、ＣＮ２０１９１１２０６６１６３、ＣＮ２０１８１１２８６２０９３（ＣＮ１１１１１８０６５Ａ）、ＣＮ２０１９１１４１８１５１８、ＣＮ２０２０１００６９３８３３、 The cell-free protein synthesis system, DNA template construction and amplification method described in CN2020101796894, CN202010269333X, CN2020102693382 and references cited therein implement the in vitro protein synthesis system of the present invention, the DNA template construction and amplification method of the present invention. can be used to

The cells from which the cell extract of the in vitro protein synthesis system is derived are not particularly limited as long as they can express the target protein in vitro. Applied to in vitro protein synthesis system from prokaryotic cell extract, eukaryotic cell extract (preferably yeast cell extract, more preferably Kluyveromyces lactis) disclosed in the prior art Exogenous protein, or endogenous protein of prokaryotic, eukaryotic cell system (preferably yeast cell system, more preferably Kluyveromyces lactis system) applied for intracellular synthesis may be can also be synthesized using the in vitro protein synthesis system of the present invention, or attempted to be synthesized using the in vitro protein synthesis system provided by the present invention.

A preferred form of said in vitro protein synthesis system is the IVTT system. The liquid after the IVTT reaction (referred to as IVTT reaction liquid) contains, in addition to the expressed target protein, reaction raw materials remaining in the IVTT system, particularly various factors derived from cell extracts (e.g., ribosomes, tRNA, translation-related enzymes, etc.). , initiation factor, elongation factor, termination factor, etc.). The IVTT reaction solution can provide the target protein for binding to the magnetic beads, and can also provide a mixed system for verifying the separation effect of the target protein.

3. A third aspect of the present invention is based on said biomagnetic microspheres provided in the first aspect of the present invention, further comprising said biotin or biotin analogue as a linking member and further comprising avidin by affinity complex binding action. Or provide biomagnetic microspheres linked to an avidin analogue.

The biomagnetic microspheres of the third aspect of the invention are also called avidin magnetic microspheres or avidin magnetic beads.

Said avidin or avidin analogue can not only serve as a purification medium, but can also be linked with other types of purification media as a linking element. An affinity complex binding is now formed between said biotin or biotin analogue and said avidin or avidin analogue.

In some preferred forms, the biomagnetic microspheres provided in the first aspect of the invention further comprise avidin binding to the biotin. An affinity complex binding is now formed between biotin and avidin. That is, biotin is linked to the ends of the branched chains of the polymer of the magnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin.

In some preferred forms, the avidin is streptavidin, denatured streptavidin, streptavidin analogs, or a combination thereof.

4. A fourth aspect of the present invention is based on the biomagnetic microspheres provided in the third aspect of the present invention, further comprising an affinity protein linked to the avidin or avidin analogue. I will provide a. At this time, biotin or a biotin analogue and avidin or an avidin analogue form a binding action of an affinity complex between them as linking elements, and the affinity protein can not only serve as a purification medium, It can be a connecting element, preferably a purification medium.

The biomagnetic microspheres of the fourth aspect of the invention are also called affinity protein magnetic microspheres or affinity protein magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG.

In some preferred forms, according to the biomagnetic microspheres provided in the second aspect of the invention, the purification medium is an affinity protein, and the biomagnetic microspheres are further linked to the affinity protein. and biotin bound to said binding force, wherein the purification medium is linked to the branched ends of said polymer by a linking element, said linking element formed by biotin and avidin. including affinity complexes.

In some preferred forms, the affinity protein is Protein A, Protein G, Protein L, or denatured proteins thereof. The corresponding biomagnetic microspheres are called Protein A magnetic microspheres or Protein A magnetic beads, Protein G magnetic microspheres or Protein G magnetic beads, Protein L magnetic microspheres or Protein L magnetic beads, etc. respectively.

The biomagnetic microspheres provided in the fourth aspect of the present invention are capable of strongly binding the affinity protein to the polymer branched ends on the outer surface of the magnetic bead, using the biotin-avidin-affinity protein linking method as an example. Alternatively, synchronous release of affinity proteins can be achieved by eluting avidin (e.g. streptavidin) from the specific binding sites of biotin when the affinity proteins need to be exchanged, and new avidin-affinity proteins The activated site of covalent complex E (eg, streptavidin-tagged affinity protein) can be reattached, resulting in rapid recovery of magnetic bead purification performance and greatly reduced antibody isolation and purification costs. The process of eluting the affinity protein-modified biomagnetic microspheres, removing the avidin-affinity protein covalent complex E, and thereby recapturing the biotin-modified biomagnetic microspheres, is the process of removing biotin-modified biomagnetic microspheres. called play. The regenerated biotin-magnetic microspheres have released biotin active sites and can rebind the avidin-affinity protein covalent complex E, again yielding affinity protein-modified biomagnetic microspheres F (biomagnetic (corresponding to regeneration of microspheres F) can be obtained to provide fresh affinity proteins and provide new antibody binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated and used, ie the affinity protein can be exchanged and reused.

5. A fifth aspect of the invention provides a method of making the biomagnetic microspheres provided in the first aspect of the invention.

5.1. Manufacture and Principles of the Biomagnetic Microspheres Provided in the First Aspect Provided in the first aspect of the invention are biotin magnetic microspheres modified with biotin or a biotin analogue.

Take for example the case where biotin is modified.
The biomagnetic microspheres provided in the first aspect provide magnetic beads (commercially available or home-made) coated with _SiO2 , activatingly modify the _SiO2 , and covalently bond a polymer to the _SiO2 (the polymer is Covalently bonded to SiO ₂ by one end of the linear backbone and a large amount of side branching distributed along the polymer backbone), biotin is covalently bonded to the branched chain ends of the polymer. By way of explanation, the above steps do not require complete isolation, allowing the integration of two or three steps into one step, e.g. activated silica-coated magnetic beads ( commercially available or homemade) can be provided directly. The step of activating and modifying SiO ₂ is, for example, step (1) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention. The step of covalently bonding the polymer to SiO ₂ is, for example, steps (2), (3) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention. The step of covalently bonding biotin to the ends of the polymer branches is, for example, step (4) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention.

The biomagnetic microspheres are produced by ( ₁ ) providing or manufacturing a magnetic microsphere body and having reactive groups _R1 on the outer surface of the magnetic microsphere body; ( ₃ ) biotin or It can be prepared by linking biotin analogues.

Taking a magnetic material coated with _SiO2 as the main body of the magnetic microspheres, the biomagnetic microspheres are: (1) providing _SiO2 coated magnetic microspheres (commercially available or self _- made); (2) performing _{a polymerization reaction on the reactive group R 1 (} for example, with acrylic acid or sodium acrylate as a monomer molecule) to form a linear backbone and ( ₃ ) binding biotin or _a biotin analogue to the functional group F1 at the end of the branched chain. and can be manufactured by At this time, the polymer covalently attached to the magnetic microsphere body has a linear backbone, one end of the linear backbone is covalently attached to the reactive group _R1 , and a large amount of side chains along the polymer backbone. Branched chains are distributed.

5.2. Exemplary Example A typical method of manufacturing the biomagnetic microspheres (see FIG. 3) includes the following steps.

Step (1): providing a magnetic microsphere body, chemically modifying the magnetic microsphere body, introducing an amino group to the outer surface of the magnetic microsphere body, forming an amino group-modified magnetic microsphere A;

In some preferred forms, a coupling agent is utilized to chemically modify the magnetic microsphere body.

In some preferred forms, the silane coupling agent is an aminated silane coupling agent.

In some preferred embodiments, the magnetic microsphere body is a magnetic material coated with _SiO2 , and a silane coupling agent is used to chemically modify the magnetic microsphere body. The silane coupling agent is, in some preferred forms, an aminated silane coupling agent.

Step (2): Covalent bonding reaction between carboxyl group and amino group to covalently bond acrylic acid molecule to the outer surface of said magnetic microsphere A, introduce carbon-carbon double bond, carbon- Magnetic microspheres B containing carbon double bonds are formed.

Step (3): Using the polymerization reaction of carbon-carbon double bonds to polymerize acrylic monomer molecules (such as sodium acrylate), the resulting acrylic polymer is a branched polymer with a linear main chain and a branched chain containing functional groups F ₁ , the polymer is covalently bonded to the outer surface of magnetic microsphere B by one end of the linear backbone to form magnetic microsphere C modified with an acrylic polymer. This step can be performed without the addition of a cross-linking agent.

See the "Nouns and Terminology" section for definitions of the functional groups of the acrylic monomer molecules and polymer branches.

In some preferred forms, the functional group F ₁ is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a formate, ammonium, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or the functional group The above-mentioned "combination of functional groups" refers to functional groups contained in all branched chains of all polymers on the outer surface of one magnetic microsphere, and the types thereof are one or more. may be The meaning of "combination of functional groups" defined in the first aspect is consistent.

In some other preferred forms, the functional group is a specific binding site.

Step (4): Biotin or a biotin analogue is covalently attached to the branched end of the polymer by means of _a functional group F1 contained in the branched chain of the polymer, and biomagnetic microspheres (biotin magnetic microspheres). In the manufactured biomagnetic microspheres, an acrylic polymer (with a polyacrylic acid backbone) provides a large amount of sites to which biotin can be attached.

5.3. Specific Embodiments Specific embodiments for producing the biotin magnetic microspheres are as follows.

Specifically, taking as an example the acrylic polymer to provide a linear main chain and multiple branches, the present invention provides the following specific embodiments. With silica-coated triiron tetroxide magnetic beads as the main body of the biomagnetic microspheres, firstly, the coupling agent 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent, A silane coupling agent, more specifically an aminated silane coupling agent) is used to chemically modify the silica-coated triiron tetroxide magnetic beads to convert amino groups to the magnetic beads. It is introduced into the outer surface to complete the activation modification on _SiO2 to obtain amino group-modified magnetic microspheres A. Subsequently, the immobilized molecule (acrylic acid molecule providing one carbon-carbon double bond and one reactive group carboxyl group) is removed from the magnetic bead using covalent bonding reaction between the carboxyl group and the amino group. Covalently bound to the surface, thereby introducing carbon-carbon double bonds to the outer surface of the magnetic beads, obtaining magnetic microspheres B containing carbon-carbon double bonds. Subsequently, an acrylic monomer molecule (for example, sodium acrylate) is polymerized using the polymerization reaction of carbon-carbon double bonds, and the polymerization product is covalently bonded to the outer surface of the magnetic beads at the same time as the polymerization reaction is performed, The linking of the polymer to _SiO2 (covalent method) is completed to obtain acrylic polymer-modified magnetic microspheres C. The immobilized molecules here are acrylic acid molecules, one immobilized molecule pulls out only one polymer molecule and at the same time only pulls out one polymer linear backbone. Taking sodium acrylate as an example of the monomer molecule, the polymerization product is sodium polyacrylate, the main chain of which is a linear polyolefin main chain, and a plurality of side branched chains COONa are shared along the main chain. The functional group that is attached and included in the branched chain is also COONa. The polymerization reaction here does not use a cross-linking agent such as N,N'-methylenebisacrylamide (CAS: 110-26-9) to avoid cross-linking of the molecular chains into a network polymer, and the cross-linking agent A linear main chain is generated in the polymerized product under the condition that is not added. When the molecular chains cross-link to each other to form a network polymer, it forms a porous structure and affects the elution efficiency of the target protein.

In some preferred forms, the amount of acrylic acid used when manufacturing the magnetic microspheres B is 0.002-20 mol/L.

In some preferred forms, the amount of sodium acrylate used when manufacturing the magnetic microspheres C is 0.53-12.76 mol/L.

The outer surface of the biomagnetic microspheres can adopt other activation modification schemes than amination. For example, the aminated biomagnetic microspheres (amino group-modified magnetic microspheres A) can be further reacted with acid anhydrides or other modifying molecules, thereby carboxylating the outer surface of the biomagnetic microspheres. Or to realize chemical modification of other activation methods.

The immobilized molecule is a small molecule immobilized by covalently bonding a polymer backbone to the outer surface of a magnetic bead. The immobilized small molecule is not particularly limited, one end of which is covalently bound to the outer surface of the magnetic bead and the other end is capable of initiating polymerization reactions including homopolymerization reactions or copolymerization reactions or polycondensation reactions, or other It suffices if the ends can be copolymerized with the ends of the linear main chain of the polymer.

The immobilized molecule may be drawn with only one polymeric linear backbone, or with two or more polymeric linear backbones, as long as it does not involve chain deposition and/or an increase in retention rate. may Preferably, one immobilizing molecule draws only one polymer molecule and draws only one polymer linear backbone.

In some preferred forms, the immobilized molecule may have only one polymer linear backbone pulled out, or two or more polymer A linear backbone may be drawn. Preferably, one immobilizing molecule draws only one polymer molecule and draws only one polymer linear backbone.

In some other preferred embodiments, the acrylic monomer molecule, which is a monomer unit for polymerization, is acrylic acid, acrylate, acrylic acid ester, methacrylic acid, methacrylate, methacrylic acid ester monomer, or any of them. may be a combination of

As another embodiment of the present invention, the acrylic polymer can be replaced with other polymers. Criteria for selection were that the polymer formed had a single linear backbone, distributed a large number of side branches along the backbone, and carried functional groups on the side branches to facilitate subsequent chemical modification. to provide a large number of functional groups to the binding sites on the outer surface of the magnetic beads by means of branched chains distributed at the side ends of the polymeric linear backbone. Examples include ε-polylysine chains, α-polylysine chains, γ-polyglutamic acid chains, polyaspartic acid chains, aspartic acid/glutamic acid copolymers, and the like.

Methods for introducing polymer molecules of other alternative forms of the above polymers to the outer surface of the biomagnetic microspheres are described below. Based on the chemical structure of the polymer substitute and the type of its side-branched active groups, select the appropriate biomagnetic microsphere outer surface activation modification method, immobilized molecule type, monomer molecule type, A reaction is performed to introduce a large number of branched chain-located active groups to the outer surface of the biomagnetic microspheres.

After covalently bonding acrylic polymer molecules (e.g., sodium polyacrylate linear chains) to the outer surface of the magnetic beads, functional groups at the ends of the branched chains provide activation sites, or biotin or biotin analogue molecules are attached. Prior to ligation, the branched functional groups of the polymer molecule can be activated as required for the reaction, rendering it reactively active and forming activated sites. 3-Propanediamine is covalently attached to the active site of the polymer branch chain (each monomer acrylic unit structure can provide one active site) to form a new functional group (amino group) followed by carboxyl A covalent amidation reaction between a group and an amino group is used to covalently attach a biotin or biotin analogue molecule to a new functional group at the branched end of the polymer, and the biotin or biotin analogue is attached to the branched chain end of the polymer. Complete covalent bonding. As an example of using biotin as a purification medium, biotin-modified biomagnetic microspheres D can be obtained, one biotin molecule can provide one specific binding site. Take for example the functional group of the polymer branch chain is COONa, in this case sodium acrylate is used as the monomer molecule, which is first subjected to carboxyl group activation before covalent reaction with 1,3-propanediamine. A conventional carboxyl group activation method, such as adding EDC.HCl and NHS, can be used.

5.3.1. Preparation of acrylic polymer-modified magnetic microspheres Preparation of magnetic microsphere A: For aqueous solution of triiron tetroxide magnetic microspheres coated with silica, wash the magnetic microspheres with absolute ethanol and apply 3-aminopropyltriethoxysilane. An ethanol solution of (APTES, a coupling agent) is added, reacted, washed, and a large amount of amino groups introduced onto the outer surface of the magnetic microspheres.

Production of magnetic microsphere B: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) are added to an aqueous solution of acrylic acid to activate carboxyl groups. and added to the aqueous solution containing the magnetic microspheres A after activation. The activated carboxyl group in acrylic acid and the amino group on the outer surface of the magnetic microsphere form a covalent bond (amide bond), introducing a large amount of carbon-carbon double bonds to the outer surface of the magnetic microsphere.

Preparation of magnetic microsphere C: An aqueous solution of acrylic monomer molecules is added to magnetic microsphere B, an initiator is added, and a carbon-carbon double bond is polymerized. The carbon-carbon double bonds in the acrylic monomer molecules and the carbon-carbon double bonds on the surface of the magnetic microspheres undergo open bond polymerization, and the acrylic polymer molecules are covalently linked to the outer surface of the magnetic microspheres, where The acrylic polymer contains a carboxyl-based functional group, and the carboxyl-based functional group may exist in the form of a carboxyl group, a formate, a formate, or the like. In one preferred form, it is present in the form of sodium formate, in which case sodium acrylate or sodium methacrylate, for example, is the monomer molecule. In another preferred form, they are present in the form of formic acid esters, in which case, for example, acrylic or methacrylic acid esters are used as monomer molecules. Formates and formates can obtain relatively good reaction activity after being activated by carboxyl groups.

5.3.2. Preparation of biotin-modified biomagnetic microspheres D Solution of magnetic microspheres C: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) to prepare microspheres. After carboxyl-activating the carboxyl-based functional groups of the side branched chains of the polymer molecules on the outer surface of the sphere, an aqueous solution of propylenediamine is added to carry out a coupling reaction, and propylenediamine is added to the side-branched carboxyl groups of the acrylic polymer molecules. to convert the functional groups of the polymer side branches from carboxyl groups to amino groups.

Aqueous solution of biotin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to activate the carboxyl groups in the biotin molecule, and then added to the aqueous solution containing magnetic microspheres C, Biotin is covalently coupled to the position of the new functional group (amino group) of the side branched chain of the polymer on the outer surface of the magnetic microsphere C, and the biotin molecule is linked to each of the many side branched chains of the acrylic polymer. Biomagnetic microspheres D are obtained.

5.3.3. Preferred Examples In some preferred embodiments, the method for producing the biomagnetic microspheres D is as follows.

First, weigh out 0.5-1000 mL (20%, v/v) of an aqueous solution of silica-coated triiron tetroxide magnetic microspheres, wash the magnetic microspheres with absolute ethanol, and add 10-300 mL of 3- An ethanol solution (5% to 50%, v/v) of aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the magnetic microspheres after the washing, reacted for 2 to 72 hours, and treated with absolute ethanol. and distilled water to obtain amino group-modified magnetic microspheres A.

Take 1.0×10 ⁻⁴ to 1 mol of acrylic acid and add it to solution X of pH 4 to 6 (solution X: final concentration of 0.01 to 1 mol/L of 2-morpholinoethanesulfonic acid (CAS: 4432-31- 9), 0.1-2 mol/L NaCl aqueous solution), 0.001-0.5 mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl, CAS: 25952-53- 8) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS, CAS: 6066-82-6) are added and allowed to react for 3-60 min. The above solution was added to a pH 7.2-7.5 PBS buffer solution mixed with 0.5-50 mL of magnetic microspheres A, reacted for 1-48 hours, washed the magnetic microspheres with distilled water, and carbon- Magnetic microspheres B modified with carbon double bonds are obtained.

Take 0.5-50 mL of magnetic microball B, add 0.5-200 mL of 5%-30% (w/v) sodium acrylate solution, and add 10 μL-20 mL of 2%-20% (w/v) ) Add an ammonium persulfate solution and 1 μL to 1 mL of tetramethylethylenediamine, react for 3 to 60 minutes, and then wash the magnetic microspheres with distilled water to obtain sodium polyacrylate-modified magnetic microspheres C.

Take 0.5-50 mL of magnetic microspheres C, transfer to pH 4-6 solution X, and add 0.001-0.5 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS) are added and allowed to react for 3-60 min. Subsequently, a PBS buffer solution of pH 7.2-7.5 in which 0.0001-1 mol of 1,3-propanediamine is dissolved is added and reacted for 1-48 hours. After washing with distilled water, a PBS buffer solution was added to convert the COONa of the polymer-side branched chain in the magnetic microsphere C into an amino functional group, and add 1.0×10 ⁻⁶ to 3.0×10 ⁻⁴ mol to the solution X. of biotin was weighed out and 2.0×10 ⁻⁶ to 1.5×10 ⁻³ mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0×10 ⁻⁶ to 1 Add 5×10 ⁻³ mol of N-hydroxysuccinimide and react for 3-60 min. Subsequently, it is added to the magnetic microsphere solution after washing, reacted for 1 to 48 hours, and washed with distilled water to obtain biotin-modified magnetic microsphere D.

5.4. A third aspect of the present invention provides biomagnetic microspheres obtained by reacting the biomagnetic microspheres provided in the first aspect of the present invention with avidin or an avidin analogue.

6. A sixth aspect of the present invention provides a method of manufacturing the biomagnetic microspheres provided in the second aspect of the present invention, the method comprising: (i) providing biomagnetic microspheres according to claim 1; which can be produced in steps (1) to (4) of the fifth aspect; and connecting to the body.

In some preferred forms, the biomagnetic microspheres are manufactured by the following steps. Steps (1) to (4) are the same as in the fifth aspect, and step (5) links the purification medium to biotin at the branched ends of the polymer of said biomagnetic microspheres.

7. A seventh aspect of the present invention provides a method of manufacturing the biomagnetic microspheres provided in the second aspect of the present invention, the method comprising: (i) providing biomagnetic microspheres according to claim 1; and (ii) forming a covalent complex of avidin or an avidin analogue with a purification medium to provide the purification medium. as a raw material for binding it to the branched ends of a polymer, forming an affinity complex binding action between said biotin or biotin analogue and said avidin or avidin analogue to produce a biomagnetic microsphere with a purification medium and obtaining.

independently optionally, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing;
Optionally, this can be accomplished independently by eluting the covalent complex of the avidin or avidin analogue with the purification medium, optionally including exchanging the purification medium under suitable conditions.

In some preferred forms, the biomagnetic microspheres are manufactured by the following five steps. Steps (1)-(4) are the same as in the fifth mode, and step (5) is the same as step (ii) above.

8. An eighth aspect of the invention provides a method of making biomagnetic microspheres provided in the fourth aspect of the invention.

A fourth aspect of the invention provides affinity protein magnetic microspheres.

The biomagnetic microspheres provided in the fourth aspect of the present invention are made from the biomagnetic microspheres provided in the first aspect, and further bind a covalent complex of avidin or an analogue thereof and an affinity protein. and the affinity complexes are carried on the polymer branches of the biomagnetic microspheres by the affinity complex action between biotin or its analogues and avidin or its analogues.

In one preferred form, the biomagnetic microspheres provided in the fourth aspect of the present invention are made from the biomagnetic microspheres provided in the first aspect of the present invention, and further comprise an avidin-affinity protein covalent complex It is obtained by combining E.

avidin-affinity protein covalent complex E: also called avidin-affinity protein complex E, a complex linked in a covalent manner, with avidin or an analogue thereof at one end and an affinity protein at the other end, Both are directly linked by a covalent bond or indirectly linked by a covalent linking element. The covalent linking groups include covalent bonds, linking peptides, and the like. Avidin-affinity protein complex E includes, for example, affinity proteins with streptavidin, where affinity proteins include but are not limited to protein A, protein G and/or protein L and the like. The avidin-affinity protein complex E further includes a streptavidin-protein A complex, a streptavidin-protein A fusion protein, and a streptavidin-enhanced green fluorescent protein-protein A fusion protein (Protein A-eGFP-Streptavidin ), Protein A-eGFP-Tamavidin2, Protein A-eGFP-Tamavidin1, etc., where eGFP broadly includes eGFP variants, Streptavidin is streptavidin, and both Tamavidin1 and Tamavidin2 are avidin analogs is.

Avidin specifically binds to biotin to form an affinity complex. The binding action of the affinity complex of avidin and biotin can be replaced by the binding action of another affinity complex, and similarly the effect of reusing the affinity protein after exchange can be realized. However, the affinity complexing action provided by avidin and biotin is more preferred. This is due to the high specificity and strong affinity between the two, and biotin adds to the binding domain with avidin, further increasing the binding carboxyl group by one, and avidin is easy to produce into affinity proteins and fusion proteins. It's for.

Criteria for selection of affinity conjugates: good specificity, strong affinity, and provide one chemically conjugable site by which they can be covalently attached to the ends of polymer branches, or after being chemically modified It can be covalently attached to the branched chain ends of the polymer.

8.1. Manufacturing process In some embodiments, the following step (5) is performed based on the biomagnetic microspheres (biotin magnetic microspheres) manufactured in the fifth aspect above, and magnetic microspheres using an affinity protein as a purification medium are prepared. get the system.

Step (5): Binding a covalent complex of avidin or an avidin analogue to an affinity protein (eg avidin-affinity protein covalent complex E). A specific binding interaction between biotin or an analogue thereof and avidin or an analogue thereof binds said covalent complex (e.g. avidin-affinity protein covalent complex E) to the branched end of the polymer, biotin or An affinity complex is formed between the analogue and avidin or an analogue thereof to form affinity protein magnetic microspheres.

For example, avidin-affinity protein covalent complex E (for example, an affinity protein with streptavidin, wherein the affinity protein is selected from, but not limited to, protein A, protein G or/and protein L, etc.) , added to a system of biotin-modified biomagnetic microspheres D, exploiting the extremely strong specific affinity between biotin and avidin (e.g., streptavidin) to non-covalently attach affinity proteins to polymer branched ends on the outer surface of magnetic beads. Affinity protein-modified magnetic beads are obtained that bind and can be used for the separation and purification of antibody-based substances, using the affinity protein as a purification medium and providing a binding site for capturing the target protein.

8.2. Example of Manufacturing Process The biomagnetic microspheres provided in the fourth aspect of the present invention are exemplified by being linked to the branched ends of the polymer in the manner of biotin-avidin-affinity protein, and are manufactured by the following steps: can do.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microspheres A, and the magnetic microsphere body is coated with _SiO2 . magnetic material, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. At this time, the silane coupling agent is preferably an aminated silane coupling agent.

(2) covalent bonding of acrylic acid molecules to the outer surface of the magnetic microspheres A by utilizing the covalent bonding reaction between carboxyl groups and amino groups to introduce carbon-carbon double bonds; Magnetic microspheres B containing double bonds are formed.

(3) Polymerizing an acrylic monomer molecule (such as sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer chain is a linear main chain. and branched chains containing functional groups, the polymer is covalently bonded to the outer surface of magnetic microsphere B by one end of the linear backbone to form acrylic polymer-modified magnetic microsphere C.

A preferred embodiment of said functional group is a specific binding site.

Other preferred aspects of the functional group are consistent with the first aspect above.

(4) A biotin-modified biomagnetic microsphere D is obtained by covalently bonding biotin to the functional group contained in the branched chain of the polymer.

(5) binding the avidin-affinity protein covalent complex E to the branched end of the polymer through the specific binding action of biotin and avidin to form the binding action of the affinity complex between biotin and avidin; Affinity protein-modified biomagnetic microspheres are obtained (biomagnetic microspheres).

Optionally independently, (6) sedimenting the affinity protein magnetic microspheres with a magnet, removing the liquid phase, and washing.

Further independently optionally, (7) exchanging said avidin-affinity protein covalent complex E and eluting said avidin-affinity protein covalent complex E under suitable conditions. include.

8.3. Biomagnetic Microspheres: Avidin-Protein A conjugated biomagnetic microspheres F (Affinity protein conjugated biomagnetic microspheres F, Protein A modified biomagnetic microspheres F, Protein A modified biomagnetic microspheres F) magnetic microspheres)

Biotin-modified biomagnetic microspheres D are added to the fusion protein solution of avidin-Protein A binding complex E (eg, Protein A-eGFP-Streptavidin, Protein A-eGFP-Tamvavidin2), mixed and incubated. The specific binding of avidin (eg, Streptavidin or Tamvavidin2) and biotin anchors Protein A to the terminal groups of polymer branches on the outer surface of biomagnetic microspheres D, resulting in avidin-protein A bound biomagnetic microspheres. Get Sphere F. In the resulting biomagnetic microsphere F structure, the acrylic acid polymer side-branches contain biotin-avidin-protein A affinity complex structures, covalently linked to the polymer's linear main chain branch points by the bio-ends. to form a non-covalent strong specific binding action of the affinity complex between biotin and avidin, between avidin and protein A is covalently linked, and between avidin and protein A Fluorescent labels can be inserted, and other connecting peptides can be inserted.

Avidin-Protein A fusion proteins such as Protein A-eGFP-Streptavidin fusion protein and Protein A-eGFP-Tamvavidin2 fusion protein can be obtained by in vitro cell-free protein synthesis by IVTT reaction. At this time, the biomagnetic microsphere D is mixed with the supernatant obtained after the IVTT reaction, and affinity Accomplish protein A binding.

8.4. The amount of affinity protein binding on the outer surface of the biomagnetic microspheres can be determined by the following method (using fluorescent protein eGFP labeling as an example).

First, after the binding reaction between the affinity protein solution and the magnetic beads is completed, the biomagnetic microspheres bound with the affinity protein are adsorbed with a magnet and sedimented. The liquid phase is then separated and collected and designated as flow-through. In this case the affinity protein concentration in the liquid phase is reduced. Calculating the fluorescence intensity of the fluorescent protein eGFP bound to the biomagnetic microspheres by measuring the change in the fluorescence value of the fluorescent protein eGFP in the supernatant obtained in the IVTT reaction before and after binding to the biomagnetic microspheres, Calculate the affinity protein concentration. Adsorption of biomagnetic microspheres to affinity proteins tends to saturate when the affinity protein concentration in the flow-through is essentially unchanged compared to the affinity protein concentration in the IVTT solution prior to incubation with the biomagnetic microspheres. , meaning that the fluorescence value of the corresponding fluorescent protein eGFP does not change significantly. A pure eGFP protein is taken to establish a standard curve of fluorescence values and eGFP protein concentration, thereby determining the content, concentration of avidin-affinity protein (eg: streptavidin-protein A) bound to the biomagnetic microspheres. was measured.

When antibodies are separated and purified using protein A-modified biomagnetic microspheres, the amount of antibody binding can be calculated by the following method (using bovine serum antibodies as an example). Incubate the protein A-modified magnetic beads with a solution of bovine serum antibodies (e.g., obtain by expressing antibodies using an in vitro protein synthesis system or obtain commercially), and after the reaction is complete, add an elution buffer. A solution is used to elute the bovine serum antibodies from the magnetic beads, and the separated bovine serum antibodies are present in the eluate. The concentration of bovine serum albumin in the eluate is determined using the Bradford method. At the same time, a microplate reader test is performed using BSA as a standard protein, the protein concentration of the purified antibody can be calculated using the standard protein as a reference, and the yield of separation and purification can be calculated.

8.5. Regeneration of biomagnetic microspheres: replacement of purification media (example with protein A as affinity protein)
Protein A elution and exchange: Elution of avidin simultaneously achieves synchronous desorption of protein A, hence avidin-protein A exchange.

For example, biomagnetic microspheres F modified with protein A are added with a denaturing buffer (containing urea and sodium dodecyl sulfate), incubated in a metal bath at 95° C., and avidin- which binds biotin in biomagnetic microspheres D. Protein A fusion protein (e.g., SPA-eGFP-Tamvavidin2) is eluted to obtain regenerated biomagnetic microspheres D (which release binding sites for biotin at the ends of polymer branched chains), followed by refolded biomagnetic microspheres. A solution containing a fresh avidin-protein A fusion protein (for example, the supernatant after the IVTT reaction of SPA-eGFP-Tamvavidin2) was added to the magnetic microsphere D, and the biotin-binding site of the released biomagnetic microsphere D was added. New avidin-Protein A (eg, SPA-eGFP-Tamvavidin2) is allowed to recombine, forming a new non-covalent specific binding interaction between biotin and avidin (eg, Tamvavidin2), thereby binding Protein A. The exchange is realized to obtain the regenerated biomagnetic microspheres F.

9. Magnetic Microsphere Position Control After obtaining the biomagnetic microspheres according to the first to fourth aspects of the invention (including but not limited to biomagnetic microsphere D and biomagnetic microsphere F), the magnet is It can be used to sediment the magnetic microspheres, remove the liquid phase, and wash away adsorbed miscellaneous proteins and/or other impurities.

By controlling the size of the magnetic microspheres and the chemical and structural parameters of the polymer, said magnetic microspheres can be stably suspended in a liquid phase and will not settle out within two days or even longer. Moreover, it can be stably suspended in a liquid system under the condition that stirring is not continued. Magnetic microspheres can be controlled to a nanosize of a few microns or even submicron, while the grafting density of the polymer on the outer surface of the magnetic microsphere can be adjusted, and furthermore the hydrophilicity, structural type, hydrodynamics of the polymer itself can be controlled. Characteristics such as radius, chain length, number of branches and length of branched chains can be adjusted, which allows the magnetic microsphere system to better control the suspension performance in the system, and the magnetic microsphere system and in vitro protein Provide sufficient contact with the synthesis reaction mixture system. One preferred magnetic microsphere size is about 1 micrometer.

10. A ninth aspect of the present invention provides use of the biomagnetic microspheres according to the first to fourth aspects of the present invention in separating and purifying proteinaceous substances.

In a preferred embodiment, the biomagnetic microspheres are used for separation and purification of antibody-based substances.

The definition of said antibody-based substance refers to the term part.

The present invention particularly provides the use of said biomagnetic microspheres in the separation and purification of antibodies, antibody fragments, antibody fusion proteins and antibody fragment fusion proteins.

When the purification medium is linked to the branched ends of the polymer by a linking element comprising an affinity complex, the use further comprises recycling the biomagnetic microspheres, ie reuse after exchange of the purification medium.

11. A tenth aspect of the present invention is the use of the biomagnetic microspheres according to the second or fourth aspect of the present invention in the separation and purification of antibody-based substances, particularly antibodies, antibody fragments, antibody fusion proteins, antibody fragments. Use in the isolation and purification of fusion proteins is provided.

Said purification medium is an affinity protein.

Preferably, said affinity protein is linked to the branched chain of the polymer in a biotin-avidin-affinity protein fashion.

When said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex (e.g. the biomagnetic microspheres according to the fourth aspect), said use further regenerates biomagnetic microspheres Includes use, ie reuse after exchange of the affinity protein.

In the biomagnetic microspheres of this use, the binding action of the affinity complex resides in the branched backbone between the affinity protein and the linear backbone of the polymer, at which time it is optionally reused after exchange of the affinity protein. and the biomagnetic microspheres can be regenerated and used. Preferably, the biotin-avidin affinity complex binding action resides in the branched backbone between the affinity protein and the linear backbone of the polymer, ie the affinity protein binds to the polymer branch in a biotin-avidin-affinity protein fashion. Once ligated, the biomagnetic microspheres can also be exchanged by avidin-avidin elution at this time.

12. An eleventh aspect of the present invention provides biomagnetic microspheres. At least one type of polymer having a linear main chain and a branched chain is provided on the outer surface of the magnetic microsphere main body, one end of the linear main chain is fixed to the outer surface of the magnetic microsphere main body, and The end is free from the outer surface of the main body of the magnetic microsphere, and a purification medium is linked to the branched chain end of the polymer of the magnetic microsphere, and the purification medium is an avidin-type tag, a polypeptide-type tag, or a protein-type tag. selected from tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

In a preferred embodiment, the polypeptide tag includes a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, and a variant sequence of WRHPQFGG. any tag or variant thereof selected from a tag, a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof, said Streg tag comprising WSHPQFEK and variants thereof.

In one preferred form, the protein-based tag is any tag selected from affinity proteins, SUMO tags, GST tags, MBP tags, and combinations thereof and variants thereof.

In one preferred form, the outer surface of the main body of the magnetic microsphere has at least one type of polymer having a linear main chain and a branched chain, and one end of the main linear chain is shared on the outer surface of the main body of the magnetic microsphere. Bindingly immobilized, the other end of the polymer is free from the outer surface of the main body of the magnetic microsphere, and the affinity protein is linked to the branched chain end of the polymer of said magnetic microsphere.

Preferably, there is also a binding function of the affinity complex on the branched backbone between said affinity protein and the linear backbone of the polymer.

More preferably, said affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of Examples 2 to 6 below contains 9.78 mM pH 8.0 trishydroxymethylaminomethane hydrochloride (Tris-HCl), 80 mM acetic acid Potassium, 5 mM magnesium acetate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate; concentration is 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid , lysine, arginine and histidine, each amino acid concentration is 0.1 mM), 15 mM glucose, 320 mM maltodextrin (molar concentration is about 52 mg/mL with glucose unit calculation), 24 mM tripotassium phosphate , 2% (w/v) of polyethylene glycol 8000 (both final concentrations), and finally 50% by volume of cell extract (specifically yeast cell extract, more specifically Kluyveromyces - Add lactis cell extract).

wherein said Kluyveromyces lactis extract contains endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following manner. A modified strain based on Kluyveromyces lactis strain ATCC8585 was taken and a gene encoding T7 RNA polymerase was integrated into the genome of Kluyveromyces lactis by the method described in CN109423496A to obtain a modified strain, to which T7 RNA was added. The polymerase is expressed endogenously and the cell stock is cultured with the modified strain, followed by the production of cell extracts. The production process of Kluyveromyces lactis cell extract adopts prior art means, referring to the method described in CN109593656A. The manufacturing steps generally include the following. An appropriate amount of fermentation-cultured Kluyveromyces lactis cells can be provided as raw materials, the cells can be rapidly frozen with liquid nitrogen, the cells can be disrupted, and the supernatant can be collected by centrifugation to obtain a cell extract. The protein concentration in the obtained Kluyveromyces lactis cell extract is 20-40 mg/mL.

IVTT reaction: 15 ng/μL of DNA template (the encoded protein contains a fluorescent label) was added to the in vitro protein synthesis system, subjected to an in vitro protein synthesis reaction, uniformly mixed, and placed in a 25-30°C environment. Leave to react, reaction time is 6-18h. A protein encoded by the DNA template is synthesized to obtain an IVTT reaction containing the protein. The RFU value can be measured using the ultraviolet absorption method, and the concentration and standard curve of the RFU value can be combined to calculate the content of the protein.

Example 1 Preparation of biomagnetic microspheres (bound to biotin)
Preparation of silica-coated magnetic microspheres (also called magnetic microsphere bodies, magnetic beads, glass beads)
20 g of _Fe3O4 microspheres _were put into a mixed solvent of 310 mL of ethanol and 125 mL of ethanol, 45 mL of 28% (wt) ammonia water was added, 22.5 mL of ethyl orthosilicate was added dropwise, and stirred at room temperature for 24 h. and washed with ethanol and water after the reaction. Using triiron tetroxide microspheres with different particle sizes (about 1 μm, 10 μm, 100 μm) as raw materials, the particle size of the resulting glass beads was controlled. Said different particle size triiron tetroxide microspheres can be produced by conventional technical means.

The resulting magnetic microspheres are also called magnetic microsphere bodies because they are used as substrates for modifying purification media and connecting elements-purification media.

The resulting magnetic microspheres have a magnetic core, can be position-controlled by the action of magnetic force, and can be moved, dispersed, sedimented, etc., and are therefore broadly defined as magnetic beads.

The resulting magnetic microspheres, which have a silica coating layer, are also called glass beads, and the magnetic core contains components or fractions such as polymers, purification media, components of in vitro protein synthesis systems, nucleic acid templates, and protein expression products. Adsorption to minutes can be reduced.

From the results of several experiments, it was found that when the particle size of the magnetic microspheres was about 1 μm, the ease of suspension, the persistence of suspension, and the binding efficiency to proteins were all the highest. The IVTT reaction solution is used to provide a mixed system of target proteins, and the binding efficiency to the target protein can be increased by more than 50% when the particle size of the magnetic microspheres is about 1 μm compared to when the particle size is 10 μm, It can be increased by more than 80% compared to a particle size of 10 μm.

Biotin magnetic beads were prepared with silica-coated magnetic microspheres by the following steps.

First, 50 mL of an aqueous solution of silica-coated triiron tetroxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) is weighed out, and the solid content is 20% (v/v). to sediment the magnetic microspheres, remove the liquid phase, and wash the magnetic microspheres with 60 mL of absolute ethanol each time for a total of 5 washes. An excess of 100 mL of an ethanol solution (25%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the magnetic microspheres after the above washing and incubated in a 50° C. water bath for 48 hours. mechanically stirred for 1 hour, then mechanically stirred in a water bath at 70 °C for 2 hours, sedimented the magnetic microspheres with a magnet, removed the liquid phase, and washed the magnetic microspheres with 60 mL of absolute ethanol each time, totaling The magnetic microspheres were washed twice, followed by washing the magnetic microspheres with 60 mL of distilled water each time, and the washing was repeated three times to obtain magnetic microspheres A.

Next, 0.01 mol of acrylic acid was taken and 100 mL of solution X (solution X: 2-morpholinoethanesulfonic acid (CAS: 4432-31-9) with a final concentration of 0.1 mol / L, 0.5 mol / L NaCl aqueous solution), 0.04 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04 mol of N-hydroxysuccinimide (CAS: 6066 -82-6), stirred at room temperature to mix homogeneously, stirred to react for 15 min, adjusted the pH of the solution to 7.2 with NaHCO ₃ solid powder, and added 10 mL of the above solution after pH adjustment. Add 100 mL of PBS buffer solution to which magnetic microsphere A was added, mechanically stir in a 30° C. water bath for 20 h, sediment the magnetic microspheres with a magnet, remove the liquid phase, and wash with 60 mL of distilled water each time. The magnetic microspheres were washed, and washing was repeated six times to obtain magnetic microspheres B.

Third, take 1 mL of magnetic microspheres B, add 12 mL of 15% (w/v) sodium acrylate solution, add 450 μL of 10% ammonium persulfate solution and 45 μL of tetramethylethylenediamine, and place at room temperature for 30 minutes. Allow to react, sediment the magnetic microspheres with a magnet, remove the liquid phase, wash the magnetic microspheres with 10 mL of distilled water each time for a total of 6 washes, and magnetic microspheres C (magnetic microspheres modified with acrylic polymer). Microspheres C) were obtained.

Fourth, transfer the synthesized magnetic microspheres C into 10 mL of solution X, add 0.004 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.004 mol of N-hydroxysuccinimide, Stir at room temperature to mix evenly, stir to react for 15 min, sediment the biomagnetic microspheres with a magnet, remove the liquid phase, wash 3 times with 10 mL distilled water each time, 4.0×10 ^{− 4} mol of 1,3-propanediamine was dissolved in 10 mL of PBS buffer solution, added to the washed magnetic microspheres, mechanically stirred in a 30° C. water bath for 20 hours, and sedimented the magnetic microspheres with a magnet; Remove the liquid phase, wash 6 times with 10 mL of distilled water each time, add 10 mL of PBS buffer solution, weigh 2.5×10 ⁻⁴ mol of biotin, add 10 mL of solution X, and add 1. Add 0×10 ⁻³ mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.001 mol of N-hydroxysuccinimide, stir uniformly at room temperature, stir and react for 15 min, add NaHCO ₃ Adjust the pH of the solution to 7.2 with solid powder, add to the magnetic microspheres containing 10 mL of the PBS buffer solution washed above, mechanically stir in a 30° C. water bath for 20 hours, and remove the magnetic microspheres with a magnet. It was sedimented, the liquid phase was removed, and the biotin-modified biomagnetic microsphere D was obtained by washing ten times with 10 mL of distilled water each time.

Example 2 Preparation of Avidin-Protein A Binding Biomagnetic Microsphere F (Affinity Protein Binding Biomagnetic Microsphere F) (Protein A as Purification Medium and Biotin-Avidin Affinity Complex as Linking Element)

2.1 Synthesis of Protein A-eGFP-Avidin Fusion Protein DNA sequences composed of tripartite genes of fusion proteins Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were constructed respectively.

Here, Protein A is derived from Staphylococcus Aureus in sequence and abbreviated as SPA. The amino acid sequence of SPA has a total length of 516 amino acid residues, of which amino acids 37-327 were selected as the gene sequence used for fusion protein construction, ie, the antibody binding domain of SPA. After optimizing the sequence with an optimization program, the nucleotide sequence was obtained and identified by SEQ ID NO. : 1.

Tamvavidin2 is an avidin analogue, a protein with the ability to bind biotin. In 2009, Yamamoto et al. found that it has a very strong biotin affinity similar to streptavidin, and that it has better thermostability than streptavidin (2009_FEBS_Yamanomo T_Tamavidins -- novel avidin-like biotin-binding proteins from the Tamogitake mushroom, translation: novel avidin-like biotin-binding protein from Tamogitake).

The amino acid sequence of Tamavidin2 can be retrieved from relevant databases, for example UniProt B9A0T7, which contains a total of 141 amino acid residues, and the DNA sequence was obtained by codon conversion, optimization with an optimization program, and optimized Subsequent nucleotide sequences are for example SEQ ID NO. :2.

Further, the nucleotide sequence of the enhanced fluorescent protein eGFP according to this example is SEQ ID NO. :3 and is the A206K variant of eGFP, also called mEGFP.
Recombinant PCR method was adopted to construct DNA templates of two fusion proteins, Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, respectively. Subsequently, an in vitro cell-free protein synthesis method was adopted to obtain two fusion proteins, Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, respectively. Adopting previously disclosed in vitro cell-free protein synthesis methods, refer to patent documents CN201610868691.6, WO2018161374A1, KR20190108180A, CN108535489B, etc. SPA-eGFP-Streptavidin and SPA-eGFP-Tamavidin2 were each expressed by the IVTT system. In general, the IVTT system includes cell extracts (including genome-integrated expression RNA polymerase), DNA templates, energy systems (for example, phosphocreatine-phosphocreatine kinase system), magnesium ions, sodium ions, components such as polyethylene glycol. was added and the reaction was carried out at 28-30°C. The reaction was terminated after 8-12 hours. The resulting IVTT reaction contained a Protein A-eGFP-avidin fusion protein corresponding to the DNA template.
IVTT reaction solutions of Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were respectively obtained.

SPA-eGFP-Streptavidin (corresponding to "1" in FIG. 4) and SPA-eGFP-Tamavidin2 (corresponding to "2" in FIG. 4) were each expressed in the IVTT system, and the RFU value of the synthesized protein was measured, The results are shown in "total" in FIG.

The IVTT reaction solution of Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamavidin2 was centrifuged at 4000 rpm and 4° C. for 10 minutes, and the supernatant was retained. Labeled as IVTT supernatant.

2.2 Production of Biomagnetic Microspheres F Bound to Protein A After incubation with the magnetic microsphere D for 1 hour, the content of the fusion protein in the biomagnetic microsphere F was measured according to the method described above, and the strength of the binding ability of the two fusion proteins was compared. The results are shown in Figure 4 "supernatant".

30 μL of 10% (v/v) biomagnetic microsphere D suspension was pipetted into binding/washing buffer (10 mM Na ₂ HPO ₄ , pH 7.4, 2 mM KH ₂ PO ₄ , 140 mM NaCl, 2.6 mM KCl) was washed three times.

Take 2 mL of the IVTT supernatant containing the avidin-Protein A fusion protein, rotate and incubate with the biomagnetic microspheres D described above at 4° C. for 1 hour, and collect the unbound supernatant, i.e. the flow-through. , this step was repeated three times to obtain three different flow-throughs. That is, the same lot of Biomagnetic Microsphere D was incubated with Avidin-Protein A three times consecutively. 2 mL of the IVTT supernatant described in step 2.1 was used each time, and the corresponding flow-through obtained each time was numbered 1, 2, 3 according to the order. Fluorometric method was adopted to detect the eGFP fluorescence value of the IVTT supernatant and the three times flow-through, the results are shown in Figure 5, the excitation wavelength (Ex) is 488 nm and the emission wavelength (Em) is 507 nm. is. The close RFU values of the triplicate flow-through to the RFU values of the IVTT supernatants explain that the binding of Biomagnetic Microspheres D to Avidin-Affinity Protein A tends to saturate, and the corresponding RFU values are shown in the table below. 1.

The RFU values for SPA-eGFP-Streptavidin and SPA-eGFP-Tamvavidin2 bound to Biomagnetic Microsphere D are shown in Tables 2 and 3 below, respectively. Here, for Streptavidin, the first binding already saturates the Protein A bound amount of the biomagnetic microsphere D. After the second binding to Tamvavidin2, the amount of Protein A binding of Biomagnetic Microsphere D almost reaches saturation. The first binding amount was calculated by subtracting the protein amount in flow-through 1 from the protein amount in the supernatant, and the second binding amount was calculated by subtracting the protein amount in flow-through 2 from the supernatant protein amount. The third binding amount was calculated by subtracting the protein amount in flow-through 3 from the protein amount in the supernatant, and is shown in Tables 2 and 3 below. Here, avidity refers to Protein A fusion protein/Biomagnetic Microsphere D and is the mass-to-volume ratio in units of (mg/mL).

Calculate the protein concentration bound each time based on the standard curve of fluorescence values and protein mass concentrations, and calculate the total protein amount bound each time based on the incubation volume (2 mL).
The formula for converting the estimated RFU value of eGFP to protein concentration based on a standard curve prepared from purified eGFP is as follows.

where X is the protein mass concentration (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), N is the molecular weight of SPA-eGFP-Streptavidin (77 .3 kDa) or the molecular weight of SPA-eGFP-Tamvavidin2 (79.4 kDa).

By substituting the measured RFU fluorescence value, the mass concentration (μg/mL) of the target protein SPA-eGFP-Streptavidin or SPA-eGFP-Tamvavidin2 can be calculated.

where Y is the RFU value of the IVV supernatant, flow-through of the avidin-Protein A fusion protein; The volume of the A solution can be multiplied to give the total protein amount of the avidin-Protein A fusion protein. Subtract the amount of avidin-protein A protein in the flow-through from the amount of avidin-protein A protein in the IVTT supernatant and the resulting difference is the amount of avidin-protein A protein bound to the biomagnetic microspheres W is. W was divided by the column bed volume of the magnetic microspheres to calculate the mass of avidin-Protein A fusion protein bound to the magnetic microspheres per unit volume, ie binding force, in mg/mL.

Since 30 μL of 10% (v/v) biomagnetic microsphere D suspension was used in this example, Tables 2 and 3 were converted into binding amounts of 1 mL of 100% biomagnetic microsphere D. shown in Here, the binding force between Tamvavidin2 and biomagnetic microsphere D is slightly stronger than that of Streptavidin.

Example 3 Purification of Serum Antibodies with Biomagnetic Microspheres F Modified with Protein A Biomagnetic microspheres bound with different volumes of SPA-eGFP-Streptavidin fusion protein were rotary incubated with 1 mL of newborn bovine serum for 1 hour at 4°C. followed by 3 washes with 1 mL binding/wash buffer, each time rotating at 4° C. for 10 minutes. The antibody was eluted with 100 μL of 0.1 M pH 2.8 glycine, the eluted antibody was in the eluate, and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was added to the eluate. added immediately. A Protein A agarose column manufactured by Seiko was used as a control (catalog number C600957-0005).

40 μL of the eluate was taken, 10 μL of 5×SDS sample buffer (containing no reducing agent) was added, subjected to a metal bath at 95° C. for 10 minutes, and subjected to SDS-PAGE electrophoresis detection. The detection results are shown in FIG. .

As can be seen from FIG. 6, a 6 μl column bed volume of biomagnetic microspheres bound with SPA-eGFP-Streptavidin fusion protein can be used to obtain better separation and purification effect than commercial products.

Washing the eluted biomagnetic microspheres three times with 1 mL of binding/washing buffer and repeating the incubation with antibody, washing and elution steps described above, i.e. after the first antibody incubation, washing and eluting; Bound antibody is released, protein A-modified magnetic microspheres are obtained again, incubation with antibody again, washing, elution are performed, and protein A-modified magnetic microspheres are obtained again, followed by a third cycle. Incubate, wash, and elute at After a total of 3 incubations of the biomagnetic microspheres with the antibody, 40 μL of the antibody-containing eluate obtained after the 3rd incubation with the antibody was taken and 10 μL of 5× sample buffer (without reducing agent) was added, After a metal bath at 95° C. for 10 minutes, SDS-PAGE electrophoresis detection was performed, and the detection results are shown in FIG.

As can be seen from FIGS. 6 and 7, the SPA-eGFP-Streptavidin-bound biomagnetic microspheres F can be repeatedly utilized multiple times, and after incubation with antibody and elution of antibody, a second, three Incubated with bovine serum again for the second time, both can extract antibodies in bovine serum, and the overall effect is not inferior to Protein A agarose magnetic beads manufactured by Seiko. For a column bed volume of 6 μL, the present invention has even higher efficiency. The biomagnetic microsphere F bound with the affinity protein (here, protein A) provided in the present invention can be used repeatedly multiple times.

Example 4 Recyclability of Binding of Biomagnetic Microsphere D and Affinity Complex E (Avidin-Protein A)

4.1 Incubation Incubation preparation of biomagnetic microspheres F conjugated with SPA-eGFP-Tamvavidin2:
Take 50 μL of 10% Biomagnetic Microsphere D suspension (volume is 5 μL) and wash 3 times with 2 mL binding/washing buffer, then add 2 mL of IVTT supernatant of SPA-eGFP-Tamvavidin2 at 4°C. 1 h at 4° C., discarded supernatant (supernatant was collected to give flow-through 1), repeated addition of fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2, and rotatable incubation at 4° C. for 1 h. discard the supernatant (collect the supernatant to give flow-through 2), add fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2 for the third time, rotate and incubate at 4°C for 1 hour, The supernatant was discarded (supernatant was collected to give flow-through 3). The RFU value of the IVTT supernatant of SPA-eGFP-Tamvavidin2 and the flow-through of three times was measured, and the remaining content of SPA-eGFP-Tamvavidin2 in the solution was calculated based on the above formula, and the results are shown in FIG. 8 and Table 4. shown in

The formula for converting the estimated RFU value of eGFP to protein concentration based on a standard curve prepared from purified eGFP is as follows.

where X is the protein mass concentration (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), N is the molecular weight of SPA-eGFP-Tamvavidin2 (79 .4 kDa).

By substituting the measured RFU fluorescence value into the formula, the mass concentration (μg/mL) of the target protein SPA-eGFP-Tamvavidin2 can be obtained. For example, if the Y value is 1330.3, the corresponding X value is calculated to be 247.6 according to the above formula and is shown in Table 4.

4.2 Elution and Exchange Avidin-affinity protein complex E: SPA-eGFP-Tamvavidin2 was eluted and exchanged.

After washing the Biomagnetic Microspheres F three times with 2 mL binding/washing buffer, add 200 μL denaturing buffer (1 M Urea and 10% SDS) and place in a metal bath at 95° C. for 10 minutes to incubate. Avidin-Protein A bound to the biomagnetic microspheres was eluted. 20 μL of this eluate was taken, 5 μL of 5×SDS sample buffer was added, and SDS-PAGE electrophoresis was performed to obtain a band in lane 1 as shown in FIG. The eluted biomagnetic microspheres D were washed three times with 2 mL of binding/washing buffer to obtain the first renatured biomagnetic microspheres D, and the binding sites for biotin on the polymer branched chains on the outer surface of the magnetic microspheres were Released.

4.3 Second incubation (regeneration)
The SPA-eGFP-Tamvavidin2 incubation and the avidin-protein A elution process were repeated to achieve the first regeneration of the biomagnetic microspheres F, ie the avidin-protein A exchange for the first time. Biomagnetic Microsphere D regenerated in the first round bound to SPA-eGFP-Tamvavidin2 and the RFU values of the corresponding IVTT supernatants and triplicate flow-throughs were measured to obtain the data in Table 5. The eluted biomagnetic microsphere-D was washed with 2 mL of binding/washing buffer three times to obtain biomagnetic microsphere-D regenerated for the twelfth time.

4.4 Third Incubation (Second Regeneration)
The SPA-eGFP-Tamvavidin2 incubation and the avidin-protein A elution process were repeated again to achieve the second regeneration of the biomagnetic microspheres F, ie the avidin-protein A exchange for the second time. Biomagnetic Microsphere D, regenerated for the second time, bound SPA-eGFP-Tamvavidin2 and measured the RFU values of the corresponding IVTT supernatant and duplicate flow-throughs, yielding the data in Table 6. In Tables 4-6, avidity refers to Protein A fusion protein/Biomagnetic Microspheres D and is the mass-to-volume ratio in units (mg/mL).

The ability of renatured biomagnetic microspheres D to rebind with protein A-eGFP-avidin was measured.

Biomagnetic microsphere D obtained for the first time saturated-bound SPA-eGFP-Tamvavidin2 in the first time, and biomagnetic microsphere D regenerated in the first time saturated-bound SPA-eGFP-Tamvavidin2 in the second time. The biomagnetic microspheres D regenerated into SPA-eGFP-Tamvavidin2 were saturated bound to SPA-eGFP-Tamvavidin2 for the third time, protein was eluted with denaturation buffer, and three eluates each containing SPA-eGFP-Tamvavidin2 were collected. , SDS-PAGE was performed. The results are shown in FIG. Lanes 1, 2, 3 are biomagnetic microspheres F obtained for the first time after saturation binding of Protein A-eGFP-avidin in the first, second and third rounds, respectively, and biomagnetic microspheres F regenerated in the first round, respectively. Sphere F, representing the electrophoretic bands of the three elutions containing SPA-eGFP-Tamvavidin2 obtained by eluting proteins from the second renatured biomagnetic microsphere F;

4.5 Measurement of Bovine Serum Antibody Binding Capacity of Regenerated Biomagnetic Microspheres F Biomagnetic microspheres regenerated for the second time after saturation binding of biomagnetic microspheres D with SPA-eGFP-Tamvavidin2 for the third time. got an F. 2 mL of newborn bovine serum (commercially available, same as below) was added to the obtained biomagnetic microspheres F regenerated for the second time, and incubated with rotation at 4° C. for 1 hour, and the supernatant was discarded (supernatant was collected to obtain flow-through 1), 2 mL of newborn bovine serum was added again, incubated with rotation at 4° C. for 1 hour, and the supernatant was discarded (supernatant was collected to obtain flow-through 2). Washed 3 times with 1 mL binding/wash buffer, rotating incubation for 10 minutes at 4°C each time. Bovine serum antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine solution (antibodies bound to protein A ends on the magnetic microspheres) and 1/10 volume (i.e. 10 μL) of 1M was added to the eluted bovine serum antibody solution. A pH 8.0 Tris-HCl solution was immediately added to obtain 110 μL of eluate.

40 μL of the eluate was taken, 10 μL of 5×SDS sample buffer (no reducing agent) was added, subjected to a metal bath at 95° C. for 10 minutes, and then subjected to SDS-PAGE electrophoresis detection. The detection results are shown in FIG. As can be seen from the experimental results in FIGS. 7, 9 and Table 6, biomagnetic microsphere D has reproducible binding with SPA-eGFP-Tamvavidin2, and biomagnetic microsphere F provided in the present invention Can be used reproducibly. Also, the step of adding the denaturing solution and heat-treating does not affect the binding efficiency of the biomagnetic microspheres.

Example 5 Preparation of Biomagnetic Microspheres G with Protein A Binding (Protein G as Purification Medium and Biotin-Avidin Affinity Complex as Linking Element)

5.1 Synthesis of ProteinG-eGFP-Avidin Fusion Protein (Avidin-Affinity Protein Covalent Complex E)
A Protein G-eGFP-Tamvavidin2 fusion protein was synthesized using the method of Example 2, and an IVTT reaction solution containing the Protein G-eGFP-Tamvavidin2 fusion protein and an IVTT supernatant were sequentially produced.

First, the DNA sequence of ProteinG-eGFP-Tamvavidin2 fusion protein containing the three-stage gene sequence was constructed. Here, the nucleotide sequences of the "eGFP" segment and the "Tamvavidin2" segment are the same as in Example 2, and have SEQ ID Nos. : 3, SEQ ID No. :2. Here, the nucleotide sequence of the "Protein G" segment (SEQ ID No.: 4) was selected from the gene sequence of its antibody binding site from Streptococcus sp. group G.

Recombinant PCR method was adopted to construct the DNA template of ProteinG-eGFP-Tamvavidin2 fusion protein. In vitro amplification was performed using the RCA method. A ProteinG-eGFP-Tamvavidin2 fusion protein was synthesized using the indicated in vitro cell-free protein synthesis method (specifically employing a Kluyveromyces lactis-based cell extract).

An IVTT reaction was performed to sequentially obtain an IVTT reaction solution containing the ProteinG-eGFP-Tamvavidin2 fusion protein and an IVTT supernatant.

5.2 Preparation of Protein G-conjugated Biomagnetic Microsphere G Na ₂ HPO ₄ , pH 7.4, 2 mM KH ₂ PO ₄ , 140 mM NaCl, 2.6 mM KCl) three times.

IVTT supernatant containing 2 mL of ProteinG-eGFP-Tamvavidin2 fusion protein was taken and incubated with the biomagnetic microsphere D at 4° C. for 1 hour by rotation. Remaining fusion protein not bound to microspheres was included, and this step was repeated three times, each time incubating fresh IVTT supernatant with Biomagnetic Microsphere D, resulting in three different flow-throughs. That is, the same lot of biomagnetic microsphere D was sequentially incubated three times with the ProteinG-eGFP-Tamvavidin2 fusion protein in the IVTT supernatant. Using 2 mL of the IVTT supernatant obtained in step 5.1 each time, the corresponding three obtained flow-throughs were labeled sequentially as flow-through 1 (FT1), flow-through 2 (FT2), flow-through 3 (FT3). . Fluorometric method was adopted to detect the eGFP fluorescence value (measured by RFU value) of the IVTT supernatant and the three times flow-through, and the results are shown in FIG.

The fact that the flow-through RFU values are close to the IVTT supernatant RFU values explains the tendency of biomagnetic microsphere D to saturate binding to the ProteinG-eGFP-Tamvavidin2 fusion protein, and the corresponding RFU values are shown in Table 7 below. shown in The first binding amount was calculated by subtracting the protein amount in flow-through 1 from the protein amount in the supernatant, and the second binding amount was calculated by subtracting the protein amount in flow-through 2 from the supernatant protein amount. The third binding amount was calculated by subtracting the protein amount in flow-through 3 from the protein amount in the supernatant. The concentration of the ProteinG-eGFP-Tamvavidin2 fusion protein was calculated using the method of Example 2 using the following formula.

where X is the mass concentration of ProteinG-eGFP-Tamvavidin2 fusion protein (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), and N is ProteinG-eGFP - is the molecular weight of the Tamvavidin2 fusion protein.

Here, the binding force is the mass of the Protein G fusion protein bound to the magnetic beads and the volume of the magnetic beads used.

By the process of incubating the biomagnetic microspheres D and the IVTT supernatant containing the Protein G fusion protein, biomagnetic microspheres G to which Protein G is bound by an affinity complex (biotin-avidin) linkage method are obtained. Also referred to as magnetic beads.

5.3. Protein G magnetic bead-purified serum antibody (targeting IgG antibody)
Protein G magnetic beads bound with different volumes of ProteinG-eGFP-Tamvavidin2 fusion protein were rotary incubated with 1 mL of newborn calf serum for 1 hour at 4° C., followed by washing with 1 mL of binding/washing buffer (three times). , rotated for 10 min each time at 4°C. Antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was added immediately into the eluate. FIG. 11 shows the results of measuring the IgG antibody purity in the eluate by SDS-PAGE. Quantitative analysis is performed based on grayscale values and the purity is greater than 95%.

Example 6 Production of biomagnetic microsphere H bound with nanoantibody anti-eGFP (using nanoantibody anti-eGFP as purification medium and biotin-avidin affinity complex as linking element)

6.1. Synthesis of antiEGFP-mScarlet-Avidin fusion protein (antiEGFP-mScarlet-Tamvavidin2 fusion protein, Nanobody fusion protein) Using the method of Example 2, an antiEGFP-mScarlet-Tamvavidin2 fusion protein was synthesized (abbreviated as antiEGFP fusion protein). , molecular weight 59 kDa), an IVTT reaction solution containing the antiEGFP-mScarlet-Tamvavidin2 fusion protein, and an IVTT supernatant were prepared in sequence.

First, the DNA sequence of the antiEGFP-mScarlet-Tamvavidin2 fusion protein containing the triple gene sequence was constructed.

Here, the nucleotide sequence of the "Tamvavidin2" segment is the same as in Example 2, SEQSEQ ID No. :2.

Here, antiEGFP has an amino acid sequence of, for example, SEQ ID No. : Nanobody shown in 5.

Here mScarlet is a bright red fluorescent protein and the corresponding nucleotide sequence is SEQ ID No. :6.

Recombinant PCR method was adopted to construct the DNA template of antiEGFP-mScarlet-Tamvavidin2 fusion protein. In vitro amplification was performed using the RCA method. The antiEGFP-mScarlet-Tamvavidin2 fusion protein was synthesized using the in vitro cell-free protein synthesis method indicated above.

An IVTT reaction was performed to sequentially obtain an IVTT reaction solution containing the antiEGFP-mScarlet-Tamvavidin2 fusion protein and an IVTT supernatant.

6.2 Preparation of Nanobody anti-eGFP-conjugated Biomagnetic Microspheres H Pipette 30 μL of 10% (v/v) Biomagnetic Microspheres D obtained in Example 1 and add binding/washing buffer ( 10 mM _Na2HPO4 , pH 7.4, ₂ mM _KH2PO4 , 140 mM NaCl, 2.6 mM KCl) was washed _three times.

Take 2 mL of IVTT supernatant containing antiEGFP-mScarlet-Tamvavidin2 fusion protein (RFU value 2400), rotate and incubate with the biomagnetic microsphere D at 4 ° C. for 1 hour, collect the supernatant, which is the flow-through (RFU value 1700). The flow-through contained residual fusion protein not bound to microspheres. The conditions for measuring the RFU value were an excitation wavelength (Ex) of 569 nm and an emission wavelength (Em) of 593 nm.

Through the process of incubating the biomagnetic microspheres D and the IVTT supernatant containing the antiEGFP fusion protein, the incubated magnetic beads are bound to the nanoantibody anti-eGFP by affinity complex (biotin-Tamvavidin2) linkage, It is written as biomagnetic microsphere H, and also written as antiEGFP magnetic beads (nanoantibody magnetic beads).

6.3. Measurement of loading amount of antiEGFP magnetic beads that binds to eGFP protein (eGFP is the target protein, excess eGFP)
30 μL of 10% (w/v) of this Example 6.2. The anti-EGFP magnetic beads prepared in were pipetted and washed three times with binding/wash buffer and ready for use.

2 mL of the IVTT reaction solution of the eGFP protein (the nucleotide sequence encoding eGFP in the DNA template is shown in SEQ ID No. 3) was measured and its fluorescence value was measured and recorded as the total fluorescence value. This was mixed with 3 μL of the washed anti-EGFP magnetic beads and incubated with rotation for 1 hour. Based on the fluorescence value measurement results in Table 8, the eGFP formula was used to obtain the corresponding amount of eGFP protein, and the resulting antiEGFP magnetic beads carried an amount of 17.7 mg/mL (antiEGFP magnetic per milliliter). mass of eGFP protein bound to the beads).

6.4. Purification efficiency of eGFP protein by antiEGFP magnetic beads (eGFP is the target protein, magnetic beads are excessive)
The antiEGFP magnetic beads prepared in this Example 6.2 were taken, washed three times with binding/wash buffer and ready for use.

1 mL of the eGFP protein IVTT reaction solution (nucleotide sequence encoding eGFP in the DNA template is shown in SEQ ID No. 3) was taken, and the fluorescence value was measured and indicated as Total. This was mixed with an excess of antiEGFP magnetic beads, incubated with rotation for 1 hour, the supernatant was collected, marked as Flow-through, and its fluorescence value was measured. The magnetic beads were washed twice with 1 mL of binding/washing buffer, rotated and incubated at 4° C. for 10 minutes each time, and the resulting washes were designated as Washing1 and Washing2, respectively, and the respective fluorescence values were measured. Incubated magnetic beads bound to the target protein eGFP. FIG. 12 and Table 9 show the measurement results of the above fluorescence values. The results show that the anti-eGFP magnetic beads obtained in this scheme can effectively bind eGFP protein and elute it. Based on the fluorescence values of the IVTT supernatant and flow-through, the binding efficiency of the antiEGFP magnetic beads to the target protein eGFP was calculated to be 98.2% after the 1 hour incubation.

Antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine eluate and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was immediately added into the eluate. The fluorescence value of the eluate was measured, described as Elution, and the purity was examined by SDS-PAGE. As shown in FIG. 13, the purity was about 95%.

In addition, what has been described above is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the above-described embodiments. A person skilled in the art can make various changes and modifications within the conceptual scope of the technical solution of the present invention or under the guidance and suggestion thereof, and any changes and modifications with equivalent technical effects made , are all within the protection scope of the present invention.

This application claims priority from Chinese Patent Application No. 201911209156X with filing date of November 30, 2019 and Chinese Patent Application No. 2020103469030 with filing date of April 28, 2020. The present application cites the full text of the above Chinese patent application.

TECHNICAL FIELD The present invention belongs to the technical field of biochemistry, and specifically relates to biomagnetic microspheres and methods for their preparation and use.

Separation and purification of proteins (including but not limited to antibodies, antibody fragments or fusion proteins thereof) is an important downstream step in the biopharmaceutical manufacturing process, and the efficiency and efficiency of separation and purification affect the quality and production cost of protein drugs. directly affect. Materials such as agarose gels are commonly used as purification columns and purification microsphere carriers for protein purification. In the prior art, the separation and purification of antibody-based substances (e.g., antibody molecules, antibody fragments, or fusion proteins thereof) are mainly performed by production engineers using protein A (abbreviation: Protein A) affinity adsorption columns to extract antibodies in fermentation solutions or reaction solutions. Separate and purify molecules. In the protein A column, the protein A immobilized on the carrier specifically binds to the specific site at the Fc end of the antibody molecule, thereby realizing specific and efficient separation of the antibody from the solution. Materials such as agarose gel are also mainly used as carriers in protein A columns that are generally used at present.

The three-dimensional porous structure of the gel-based material is advantageous in increasing the specific surface area of the material, thereby increasing the sites that can bind purification media (e.g., immobilized protein A) and target proteins (including antibodies). increase the amount of specific binding to The three-dimensional porous structure of the carrier material can greatly improve the number of protein (including antibody) binding sites, but the porous structure inside the carrier increases the retention time of the protein when the protein elutes, and the carrier Some discontinuous spaces or blind spots inside prevent proteins from eluting from inside the material and increase the retention rate. Immobilization of the protein-binding site only on the outer surface of the carrier can avoid the protein product from entering the interior of the material and can greatly reduce the retention time and retention rate of the protein during elution. If only the outer surface of the carrier is used, the specific surface area of the carrier is greatly reduced, the number of protein binding sites is greatly reduced, and the purification efficiency is reduced.

Polymers are macromolecular compounds and can be formed by polymerizing monomer molecules. Polymerization is carried out using monomer molecules with active sites, and the polymerized product can contain a large number of active sites, greatly increasing the number of active sites, and these active sites provide additional corresponding binding sites. can be formed or introduced. Polymers have various types and structures, and have a network structure formed by cross-linking molecular chains with each other, have a linear structure of a single linear molecular chain, and have a branched structure with a plurality of branched chains (e.g. structures such as branched, tree-like, comb-like, and hyperbranched structures), and polymers with different structural types are widely used in different fields, respectively.

In the prior art, purification columns used for protein separation and purification mainly use a covalent bond method to immobilize purification media. Protein A columns for purifying antibody-based substances using protein A as a purification medium are examples. Protein A columns used for separating and purifying antibodies, antibody fragments, fusion proteins thereof, etc., mainly employ a covalent bonding method. is used to immobilize protein A, and the C-terminal cysteine covalently binds protein A to the carrier. Although the covalent attachment method can ensure that the purification medium (e.g. protein A) is firmly fixed to the support, after multiple uses of the purification column (e.g. protein A column), the purification medium (e.g. protein A) The binding performance of is reduced and the purification effect is reduced. Therefore, in order to ensure relatively high purification efficiency and quality, the operator needs to replace all the fillers in the affinity chromatography column. It is labor and time consuming and causes high purification costs.

The present invention provides biomagnetic microspheres, which can be used to separate and purify target substances, particularly protein-based substances (including but not limited to antibody-based proteins), and can bind target substances in high throughput. Not only can the retention rate of target substances during elution be effectively reduced, purification media (e.g. affinity proteins) can be conveniently exchanged, and it is fast, high-throughput, reusable, and reusable. and can greatly reduce the purification cost of the target substance. For example, when affinity proteins are used as purification media, purification costs for antibody-based proteins can be greatly reduced.

1. A first aspect of the present invention provides biomagnetic microspheres, said biomagnetic microspheres comprising a magnetic microsphere body, the outer surface of said magnetic microsphere body having at least one linear backbone and a polymer having a branched chain, one end of the linear main chain is fixed to the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the biomagnetic microsphere Biotin or biotin analogues are linked to the branched ends of the polymer. Said biotin or biotin analogue can not only serve as a purification medium, but can also be linked as a linking element to still other types of purification media.

Said biomagnetic microspheres are also called biotin magnetic microspheres or biotin magnetic beads.

The term "anchored to" refers to the linear backbone being "anchored to" the outer surface of the magnetic microsphere body in a covalent manner.

In addition, the linear main chain is covalently fixed to the outer surface of the magnetic microsphere body in a direct manner, or shared on the outer surface of the magnetic microsphere body in an indirect manner via a linking group (linking element). Cohesively fixed.

Furthermore, the number of branched chains in the polymer is plural, preferably at least three.
In one preferred form, the size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 600 μm, 600 μm, 650 μm, Any one particle size scale of 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm or a range between any two particle size scales, said diameter size being an average value.

In one preferred form, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.2 to 6 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.4 to 5 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.5 to 3 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.2 to 1 μm.

In one preferred form, the diameter of the magnetic microsphere bodies is selected from 0.5 to 1 μm.

In one preferred form, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In one preferred form, the magnetic microsphere body has a diameter of 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, The deviation is ±20%, more preferably ±10%.

In one preferred form, the backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone. See the "Nouns and Terminology" section for definitions of said acrylic polymers. In one preferred form, the polyolefin backbone is at the same time an acrylic polymer backbone (ie the linear backbone of the polymer is both a polyolefin backbone and provided from an acrylic polymer backbone).

In a preferred embodiment, the monomer unit of the acrylic polymer is preferably any one of acrylic monomer molecules such as acrylic acid, acrylate, acrylic acid ester, methacrylic acid, methacrylate, and methacrylic acid ester, or selected from a combination thereof. Said acrylic polymer may be obtained by polymerizing any of the above monomers, or may be obtained by copolymerizing the corresponding combination of the above monomers.

In one preferred form, the branched chains of the polymer are covalently attached to biotin or biotin analogues by functional group-based covalent bonds, covalently attaching biotin or biotin analogues to the branched ends of the polymer. It can be obtained by covalent reaction of biotin or biotin analogues with functional groups contained in branched chains of polymer molecules on the outer surface of biomagnetic microspheres. Here, one preferred embodiment of said functional group is a specific binding site (see "Nouns and Terms" section of specific embodiments for definition).

The covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group. Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. A preferred salt form of the carboxyl group is a sodium salt form, for example, COONa, and a preferred salt form of the amino group may be an inorganic salt form or an organic salt form. Often include, but are not limited to, hydrochloride, hydrofluoride, and like forms. The "combination of functional groups" refers to all branched chains of all polymer molecules on the outer surface of one magnetic microsphere, which can form covalent bonds based on the involvement of different functional groups. Taking biotin as an example, all biotin molecules on the outer surface of one biotin magnetic microsphere can covalently bond with different functional groups, but one biotin molecule can only link with one functional group. .

In one preferred form, the linear backbone of the polymer is covalently attached to the outer surface of the magnetic microsphere body either directly or indirectly via a linking group.

In one preferred form, said magnetic microsphere body is a magnetic material coated with _SiO2 . Alternatively, _SiO2 may itself be a silane coupling agent with active sites.

In one preferred form, the magnetic material is selected from iron oxides, iron compounds, iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, or a combination thereof.

Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNiMo), FeSi , FeAl, _FeSiAl , _MO.6Fe2O3 , GdO, or a combination thereof, wherein Re is a rare earth element and M is Ba, Sr, Pb, i.e., _{The MO.6Fe2O3} is _{BaO.6Fe2O3 , SrO.6Fe2O3 or PbO.6Fe2O3 .}

2. A second aspect of the present invention provides biomagnetic microspheres, and based on said biomagnetic microspheres provided in the first aspect of the present invention, said biotin or biotin analogue is further added as a linking element to a purification medium. are connected. That is, the branched ends of the polymer are linked to the purification medium via a linking element, and the linking element comprises the biotin or biotin analogue.

The purification media may contain, but are not limited to, avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and forms an affinity complex binding action with the avidin.

The avidin may be, but is not limited to, streptavidin, modified streptavidin, streptavidin analogs, or combinations thereof.

In a preferred embodiment, the polypeptide tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, and a tag containing a WRHPQFGG (SEQ ID No: 7) sequence. , a tag containing a variant sequence of WRHPQFGG (SEQ ID No: 7) , a tag containing a sequence of RKAAVSHW (SEQ ID No: 8) , a tag containing a variant sequence of RKAAVSHW (SEQ ID No: 8) , or a combination thereof or one of the tags or variants thereof. The Streg tags include WSHPQFEK (SEQ ID No: 9) and variants thereof.

In one preferred form, said protein type tag is selected from affinity proteins, SUMO tags, GST tags, MBP tags and any combination thereof or variants thereof.

In one preferred form, the affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

In a preferred embodiment, the antibody-based tag is an antibody, antibody fragment, antibody single chain, single chain fragment, antibody fusion protein, antibody fragment fusion protein, derivative of any, or any of is a variant of

In one preferred form, the antibody-based tag is an anti-protein antibody.
In one preferred form, the antibody-based tag is an anti-fluorescent protein antibody.
In one preferred form, the antibody-based tag is a nanobody.
In one preferred form, the antibody-based tag is an anti-protein nanobody.
In one preferred form, the antibody-based tag is an anti-fluorescent protein nanobody.
In one preferred form, the antibody-based tag is an anti-green fluorescent protein or variant nanobody.
In one preferred form, the antibody-based tag is an Fc fragment.

Modes of linkage between the purification medium and the biotin or biotin analog include, but are not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or combinations thereof.

In one preferred form, the covalent bond is a dynamic covalent bond, more preferably the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In one preferred form, said supramolecular interactions are coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions, or combinations thereof.

In one preferred form of the biomagnetic microspheres, the purification media are linked to the branched ends of the polymer by linking elements comprising affinity complexes.

In one preferred form, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In one preferred form, said biotin or biotin analogue is bound to avidin or an avidin analogue by affinity complexing and said purification medium is bound directly or indirectly to said avidin or avidin analogue.

In one preferred form, the purification medium is linked to biotin or a biotin analogue at the branched end of the polymer by an avidin-type tag-purification medium covalent complex, and is linked to the biotin or biotin analogue by an avidin-type tag. form the linking member of the affinity complex, and in a further preferred form, the purification medium binds biotin or a biotin analog at the branched chain end of the polymer to the affinity complex by means of an avidin-purification medium covalent complex. Form a connecting element.

3. A third aspect of the invention provides biomagnetic microspheres, based on said biomagnetic microspheres provided by the first aspect of the invention, further comprising said biotin or biotin analogue as a linking member, further comprising: It is linked to avidin or an avidin analogue by an affinity complex binding activity.

Said biomagnetic microspheres are also called avidin magnetic microspheres or avidin magnetic beads.

Said avidin or avidin analogue can not only serve as a purification medium, but can also be linked with other types of purification media as a linking element. An affinity complex binding is now formed between said biotin or biotin analogue and said avidin or avidin analogue.

In one preferred form, based on said biomagnetic microspheres provided in the first aspect of the invention, it further comprises avidin binding to said biotin. An affinity complex binding is now formed between biotin and avidin. That is, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and forms an affinity complex with the avidin.

In one preferred form, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof.

4. A fourth aspect of the invention provides biomagnetic microspheres, based on said biomagnetic microspheres provided in the third aspect of the invention, further comprising affinity linked to said avidin or avidin analogue. Contains protein. At this time, biotin or a biotin analogue and avidin or an avidin analogue form a binding action of an affinity complex between them as linking elements, and the affinity protein can not only serve as a purification medium, It can also be a connecting element, preferably a purification medium.

Said biomagnetic microspheres are also called affinity protein magnetic microspheres or affinity protein magnetic beads.

In one preferred form, based on said biomagnetic microspheres provided in the second aspect of the present invention, said purification medium is an affinity protein, said biomagnetic microspheres further comprising avidin linked to said affinity protein. and biotin attached to said avidin, wherein said biotin is linked to said branch of said polymer, wherein said purification medium is linked to said branch of said polymer by a linking element, and contains the affinity complex formed by biotin and avidin.

In a preferred embodiment, the affinity protein is any one of protein A, protein G, protein L, or denatured proteins thereof.

5. A fifth aspect of the invention provides a method (see FIG. 3) of manufacturing said biomagnetic microspheres provided in the first aspect of the invention, said method comprising the following steps.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microspheres A, and the magnetic microsphere body is made of _SiO2 . In the case of coated magnetic materials, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. Said silane coupling agent is preferably an aminated silane coupling agent.

(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , to form magnetic microspheres B containing carbon-carbon double bonds.

(3) Polymerizing an acrylic monomer molecule (for example, sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer has a linear main chain. and a branched chain containing a functional group, the polymer is covalently bonded to the outer surface of the magnetic microsphere B by one end of the linear backbone to form the acrylic polymer-modified magnetic microsphere C.

See the "Nouns and Terminology" section for definitions of the functional groups of the acrylic monomer molecules and polymer branches.

Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a formate, ammonium, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. The "combination of functional groups" refers to functional groups contained in all branched chains of all polymers on the outer surface of one magnetic microsphere, and the types thereof may be one or more. . It matches the meaning of "combination of functional groups" defined in the first mode.

Also preferably, said functional group is a specific binding site.

(4) biomagnetic microspheres D attached to biotin or biotin analogues by covalently coupling biotin or biotin analogues to the branched ends of the polymer via functional groups contained in the branched chains of the polymer; (biotin magnetic microspheres) are obtained.

6. A sixth aspect of the present invention provides a method of manufacturing said biomagnetic microspheres provided in the second aspect of the present invention, said method comprising the following steps.

(i) providing the biomagnetic microspheres of claim 1, which can be manufactured in steps (1) to (4) of the fifth aspect;

(ii) Linking a purification medium to biotin or a biotin analogue at the branched end of the polymer of said biomagnetic microspheres to obtain biomagnetic microspheres with bound purification medium.

7. A seventh aspect of the present invention provides a method of manufacturing said biomagnetic microspheres provided in the second aspect of the present invention, said method comprising the following steps.

(i) providing the biomagnetic microspheres of claim 1, which can be manufactured in steps (1) to (4) of the fifth aspect;

(ii) a covalent complex of an avidin or avidin analogue with a purification medium (e.g., an avidin-purification medium covalent complex) as a starting material for providing a purification medium, attached to the branched ends of a polymer, said forming an affinity complex binding action between biotin or a biotin analogue and said avidin or avidin analogue to obtain biomagnetic microspheres with a purification medium.

Optionally independently, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing.

Optionally, independently, it includes exchanging the purification medium, which can be achieved by exchanging the covalent complexes of the avidin or avidin analogue with the purification medium.

8. An eighth aspect of the invention provides a method of manufacturing the biomagnetic microspheres provided in the fourth aspect of the invention (see FIG. 3 for an example), the method comprising the following steps.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microspheres A, and the magnetic microsphere body is made of _SiO2 . In the case of coated magnetic materials, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. At this time, the silane coupling agent is preferably an aminated silane coupling agent.

(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , to form magnetic microspheres B containing carbon-carbon double bonds.

(3) Polymerize acrylic monomer molecules (for example, sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer has a linear main chain and The polymer is covalently bonded to the outer surface of the magnetic microsphere B by one end of the linear backbone to form the acrylic polymer-modified magnetic microsphere C.

A preferred embodiment of said functional group is a specific binding site.

Other preferred forms of said functional groups are consistent with the first aspect above.

(4) Biotin is covalently coupled to the functional groups contained in the branched chains of the polymer to obtain biotin-modified biomagnetic microspheres D (biotin magnetic microspheres).

(5) binding the avidin-affinity protein covalent complex E to the branched end of the polymer through the specific binding action of biotin and avidin to form the binding action of the affinity complex between biotin and avidin; , to obtain biomagnetic microspheres F (said biomagnetic microspheres F are affinity protein magnetic microspheres because they bind to affinity proteins).

Independently and optionally, (6) sedimenting the biomagnetic microspheres F with a magnet, removing the liquid phase, and washing.

Further independently optionally, (7) exchanging said avidin-affinity protein covalent complex E.

9. A ninth aspect of the invention provides the use of said biomagnetic microspheres according to the first to fourth aspects of the invention in the separation and purification of proteinaceous substances.

A preferred embodiment is the use of the biomagnetic microspheres in the separation and purification of antibody-based substances.

The antibody-based substances refer to proteins of antibodies and antibody fragments, including, but not limited to, antibodies, antibody fragments, antibody fusion proteins, and antibody fragment fusion proteins.

When the purification medium is linked to the branched ends of the polymer by a linking element comprising an affinity complex, the use further optionally comprises the recycle use of the biomagnetic microspheres, i.e. reuse after exchange of the purification medium. including.

10. A tenth aspect of the invention is the use according to the first to fourth aspects of the invention in the separation and purification of antibody-based substances of biomagnetic microspheres, in particular antibodies, antibody fragments, antibody fusion proteins, antibody fragment fusions. Use in protein separation and purification is provided.

Said purification medium is an affinity protein.

In one preferred form, the affinity protein is linked to the polymer branches in a biotin-avidin-affinity protein fashion.

Where said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex (e.g. biomagnetic microspheres according to the fourth aspect), said use further optionally comprises biomagnetic microspheres. Includes recycling of spheres, ie recycling after exchange of affinity proteins.

11. An eleventh aspect of the present invention provides biomagnetic microspheres. The outer surface of the magnetic microsphere main body has at least one type of polymer having a linear main chain and a branched chain, and one end of the linear main chain is fixed to the outer surface of the magnetic microsphere main body. The other end is free from the outer surface of the magnetic microsphere main body, and a purification medium is linked to the branched chain end of the polymer of the magnetic microsphere, and the purification medium is an avidin-type tag, a polypeptide-type tag, or a protein. A type tag, an antibody type tag, an antigen type tag, or a combination thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

In a preferred embodiment, the polypeptide tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, and a tag containing a WRHPQFGG (SEQ ID No: 7) sequence. , a tag containing a variant sequence of WRHPQFGG (SEQ ID No: 7) , a tag containing a sequence of RKAAVSHW (SEQ ID No: 8) , a tag containing a variant sequence of RKAAVSHW (SEQ ID No: 8) , or a combination thereof and its variants. The Streg tag comprises WSHPQFEK (SEQ ID No: 9) or variants thereof.

In one preferred form, said protein type tag is selected from any of affinity proteins, SUMO tags, GST tags, MBP tags and combinations thereof or variants thereof.

In a preferred embodiment, the outer surface of the main body of the magnetic microsphere has at least one type of polymer having a linear main chain and a branched chain, and one end of the main linear chain is attached to the outer surface of the main body of the magnetic microsphere. Covalently immobilized, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the affinity protein is linked to the branched chain end of the polymer of said magnetic microsphere.

Preferably, there is also a binding function of the affinity complex on the branched backbone between said affinity protein and the linear backbone of the polymer.

More preferably, said affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

The main advantages and positive effects of the invention include the following.

One of the core of the present invention is the structure of the biomagnetic microspheres, in which linear backbone-containing polymer molecules are covalently anchored to the outer surface of the magnetic microsphere body, and these polymer molecules have an additional large number of functions. functionalized branched chain to which purification media (including but not limited to biotin, avidin, affinity proteins, polypeptide tags, protein tags, etc.) are linked. The above structure not only provides a purification medium suspended on the side ends of a large amount of polymeric linear backbones on the outer surface of the biomagnetic microspheres to avoid the high retention rate caused by conventional network structures, but also has a specific surface area of can provide a large number of target substance (eg, antibody) binding sites, overcoming the limitations of . Here, the type of purification medium can be selected according to the type of substance to be purified. In addition, if the purification medium is in the form of an affinity complex and is linked to the branched chains of the polymer by strong non-covalent interactions, a branched backbone between the purification medium (e.g. affinity protein) and the polymer linear backbone There may also be a binding activity of the affinity complex in , whereby the purification medium (eg parent protein) can be readily exchanged. When the target substance to be separated and purified is an antibody-based protein, the purification medium is usually an affinity protein. The description of the manufacturing and principle part of the magnetic microspheres should be combined and understood.

Further, the core of the present invention lies in the construction process (manufacturing method) of the biomagnetic microsphere structure described above, in which a plurality of binding sites are formed on the outer surface of the magnetic beads (the outer surface of the main body of the biomagnetic microsphere) by chemically modifying the outer surface. followed by covalent attachment of a polymer molecule to a single binding site on the outer surface of the magnetic bead, the polymer molecule being covalently attached by one end of the linear backbone to the single binding site on the outer surface of the magnetic bead. , a large number of side-branched chains are distributed along the linear main chain, and the side-branched chains carry the nascent binding sites, thereby multiplying the binding sites by multiples, tens, hundreds, hundreds, or even thousands of times. Furthermore, based on specific purification requirements, specific purification media can be linked to the nascent binding sites of polymer branches to generate corresponding specific target molecules (especially biochemical molecules, antibody-based protein molecules). (including but not limited to) capture. Also, a single binding site of a biomagnetic microsphere can covalently bind not only to one linear polymer backbone, but also to two or more linear backbones. , preferably does not cause chain build-up and increase the retention rate.

Preferably, a single binding site can pull only one linear backbone, thus providing the linear backbone with a large active space.

Also preferably, one binding site pulls only two linear backbones, providing as much activity space for the linear backbones as possible.

The main advantages and positive effects of the present invention further include:
(1) The structural design of the present invention coats the magnetic microsphere surface with a polymer carrying a large amount of branched special structures to overcome the limitation of specific surface area to provide a large amount of purification media binding sites and to The number of purification media that can be bound to the surface of the sphere has been expanded several times, ten times, several tens of times, hundreds of times, hundreds of times, and even a thousand times, and high-throughput binding to target substances has been achieved. The substance is a proteinaceous substance. Biomagnetic microspheres efficiently capture target substances (e.g., target proteins, including but not limited to antibodies, antibody fragments, or fusion proteins thereof) from mixed systems onto the magnetic microspheres for high-throughput binding. to achieve high-throughput separation.

(2) The flexibility of the polymer chain itself can be used, and the polymer chain can flexibly oscillate in the reaction purification mixing system, expanding the activity space of the purification medium and increasing the capture rate and binding amount for proteins. promote rapid and sufficient binding of the target substance, realizing high efficiency and high throughput.

(3) The structural design of the present invention affects the efficiency of purified target substances (e.g., target proteins, including but not limited to antibodies, antibody fragments, or fusion proteins thereof) when biomagnetic microspheres elute. It can achieve effective elution, effectively reduce the retention time and retention rate of the target substance, and achieve high efficiency and high yield. The purification medium can be linked to the branched chain ends of the polymer, and in one aspect, the structure of the polymer does not form a network structure, does not cause accumulation of branches, and avoids the occurrence of discontinuous spaces and blind spots. , avoiding the high residence time and high residence fraction of conventional reticulated structures. On the other hand, the branched chains of the polymer further act as spatial spacing, allowing the purification media to be well distributed in the mixed system, away from the magnetic microsphere surface and the internal backbone of the polymer, increasing the efficiency of target substance capture. In addition, it effectively reduces the retention time and retention rate of the target substance during the subsequent elution step, achieving high throughput, high efficiency and high percentage separation. The structural design of the present invention not only can take advantage of the high flexibility of the linear backbone, but also combines the advantages of high-fold amplification of the number of branches, resulting in high-rate, high-throughput binding, high efficiency, and high ratio. (high yield) separation is better achieved.

(4) Purification media (e.g., affinity proteins) of the biomagnetic microspheres of the present invention are linked to the polymer branched ends on the outer surface of the magnetic beads by a strong non-covalent binding force in the manner of affinity complexes, and the purification media (e.g., When the affinity protein) needs to be renewed or replaced, the purification medium can be easily and quickly eluted from the microspheres and recombined with new purification medium, quickly restoring the purification performance of the magnetic microspheres, Biomagnetic microspheres can be regenerated and used multiple times, thereby reducing separation and purification costs.

(5) The biomagnetic microspheres of the present invention are easy to manipulate and use. When the magnetic microspheres bound to the target substance are separated from the system, it is easy to operate, and the aggregation state and position of the magnetic microspheres can be efficiently manipulated simply by using a small magnet. Realizes high-speed dispersion or high-speed sedimentation to make the separation and purification of target substances (such as antibodies) simple and fast, without the need to use large-scale experimental equipment such as high-speed centrifuges, greatly reducing the cost of separation and purification. Reduce.

(6) The biomagnetic microspheres provided by the present invention are versatile and have selectable purification media. Depending on the type of specific purification substrate, the magnetic microsphere system can be equipped with flexible purification media to achieve the capture of specific target molecules (especially proteinaceous substances). For example, affinity proteins are targeted and generally used for large-scale separation and purification of antibody-based substances, including, but not limited to, antibodies, antibody fragments, or fusion proteins thereof.

1 is a structural schematic diagram of a biomagnetic microsphere provided in the first aspect of the present invention; FIG. Here, the magnetic microsphere body is exemplified by Fe ₃ O ₄ coated with SiO ₂ and biotin is shown as the purification medium. In the figure, the number of polymer molecules (4) is for simplicity and schematic only, and does not mean that the number of polymer molecules on the outer surface of the magnetic microspheres is limited to 4. can be controlled and adjusted based on the content of each raw material in Similarly, the number of branches pendant to the linear main chain side ends is for illustrative purposes only and is not intended to limit the number of side branches in the polymer molecules of the present invention. FIG. 4 is a structural schematic diagram of a biomagnetic microsphere provided in the fourth aspect of the present invention; Protein A is shown as the purification medium and is attached to the branched ends of the brush-like structure by the “biotin-avidin-protein A scheme”. Here, the biomagnetic microsphere body is exemplified by Fe ₃ O ₄ coated with SiO ₂ . In the figure, the number of polymer molecules and the number of branched chains at the end of the main chain are merely examples, and do not limit the number of side branched chains of the polymer molecule of the present invention. Figure 2 is a flow chart of a method for manufacturing biomagnetic microspheres provided in the fourth aspect of the invention; Here, the manufacturing process from the amino group-modified magnetic microsphere A to the biomagnetic microsphere D corresponds to the manufacturing method of the biomagnetic microsphere provided in the fifth aspect. Biomagnetic Microsphere D (biotin magnetic beads) is the result of experiments before and after binding with protein A-eGFP-avidin. It was incubated once with the result of RFU value measurement. Protein A-eGFP-avidin (SPA-eGFP-avidin) was produced by an in vitro protein synthesis system, and the supernatant after IVTT reaction (abbreviated as IVTT supernatant) was obtained, and before binding with biomagnetic microsphere D and compare the changes in solution RFU values. In the two protein A-eGFP-avidins, where the avidin corresponding to number 1 is Streptavidin and the avidin corresponding to number 2 is Tamvavidin2. "Total" corresponds to the IVTT supernatant RFU value before binding (before biomagnetic microsphere treatment) and "Supernatant" corresponds to the IVTT supernatant RFU value after binding (after biomagnetic microsphere treatment). Fig. 2 shows experimental results of producing biomagnetic microspheres F (Protein A magnetic beads). This is the measurement result of the RFU value, and protein A-eGFP-avidin was saturated bound. The biomagnetic microspheres D were repeatedly combined with the solution obtained after the IVTT reaction expressing protein A-eGFP-avidin (abbreviated as IVTT supernatant) to form biomagnetic microspheres saturated with protein A-eGFP-avidin. Get Sphere F. where the avidin corresponding to 1 is Streptavidin, the avidin corresponding to 2 is Tamvavidin2, supernatant corresponds to IVTT supernatant not treated with biomagnetic microspheres, Flow-through1, Flow-through 2 (flow-through 2) and Flow-through 3 (flow-through 3) correspond to 3 cycles of elution (debinding release), followed by continuous incubation of magnetic microspheres with avidin-protein A (capture binding). , flow-throughs 1, 2 and 3 obtained sequentially using the same source and amount of IVTT supernatant each time. Biomagnetic microsphere F is an experimental result of separating and purifying an antibody, and the avidin in biomagnetic microsphere F is Streptavidin. The biomagnetic microspheres F were eluted after incubation with the antibody IgG solution, the antibody IgG was captured and separated from the antibody solution, eluted and released into the eluate, and the corresponding purified antibody-containing eluate was subjected to denaturing polyacrylamide gel electrophoresis. It is a migration (SDS-PAGE) test result. Here, lanes 1, 2, 3, and 4 correspond to antibody elution bands when the column bed volumes of SPA-magnetic microspheres were 2 microliters, 6 microliters, 18 microliters, and 54 microliters, respectively. 5 is an antibody elution band of a positive control, a commercially available Protein A agarose column of Seiko Biotechnology (Shanghai) Co., Ltd. (hereinafter referred to as Seiko) with a column volume of 7.5 microliters. where M corresponds to the Marker molecular weight mark. It is the experimental result of repeated use of biomagnetic microsphere F, and the avidin in biomagnetic microsphere F is Streptavidin. Biomagnetic microspheres F were incubated with an antibody solution (antibody binding), washed, and eluted (antibody release) three times, and the eluate was subjected to an SDS-PAGE electrophoresis test. Here, lanes 1, 2, 3, and 4 correspond to antibody elution bands when the column bed volumes of SPA-magnetic microspheres were 2 microliters, 6 microliters, 18 microliters, and 54 microliters, respectively. 5 is an antibody elution band of a positive control, a commercially available Protein A agarose column of Seiko Biotechnology (Shanghai) Co., Ltd. (hereinafter referred to as Seiko) with a column volume of 7.5 microliters. where M corresponds to the Marker molecular weight mark. Fig. 4 shows the results of regeneration experiments of biomagnetic microspheres, and the results of measurement of fluorescence relative values using protein A-eGFP-Tamvavidin2; Biomagnetic microsphere D was repeatedly bound with the solution obtained after IVTT reaction of protein A-eGFP-avidin to obtain biomagnetic microsphere F (1) saturated with protein A-eGFP-avidin. The Protein A-eGFP-avidin is subsequently eluted from the biomagnetic microspheres D, and the biomagnetic microspheres D are bound a second time with the solution obtained after the IVTT reaction of fresh Protein A-eGFP-avidin,2 A second round of biomagnetic microspheres F (2) saturated with protein A-eGFP-avidin was obtained. The above steps were repeated for the third time to obtain biomagnetic microspheres F(3) saturated with protein A-eGFP-avidin. Here, supernatant corresponds to IVTT supernatant not treated with biomagnetic microspheres, Flow-through1, Flow-through2, Flow-through3 are magnetic Flow-throughs 1, 2, 3 were obtained sequentially, microspheres were successively incubated with avidin-protein A three times, each time using the same IVTT supernatant (taken fresh). Fig. 2 shows experimental results of regenerating biomagnetic microspheres and isolating and purifying antibodies after regenerating. Protein A-eGFP-Tamvavidin2 was employed. Biomagnetic microspheres F saturated with protein A-eGFP-avidin were eluted with denaturing buffer, and the eluate containing the eluted protein A-eGFP-avidin was subjected to SDS-PAGE. Lanes 1, 2 and 3 are eluted after the first saturation binding, eluted after the second saturation binding, and eluted after the third saturation binding, respectively. A band from the eluate containing protein A-eGFP-avidin is shown. Lane 4 shows the purified antibody protein band eluted with the elution buffer after incubating the third renatured Biomagnetic Microsphere F with commercial newborn bovine serum. where M corresponds to the Marker molecular weight mark. Fig. 2 shows test results of the amount of protein G supported by biomagnetic microspheres G (protein G magnetic beads, protein G magnetic beads), and measurement results of RFU values. The IVTT supernatant of biomagnetic microsphere D and protein G-eGFP-avidin was incubated three times to obtain biomagnetic microsphere G saturated bound to protein G-eGFP-avidin. Here, the Supernatant corresponds to the IVTT supernatant not treated with biomagnetic microspheres and the corresponding flow-throughs by three incubations: FT1 (flow-through 1), FT2 (flow-through 2), FT3 (flow-through), respectively. Through 3) was obtained. It is an experimental result of antibody separation and purification using biomagnetic microsphere G (Protein G magnetic beads). The biomagnetic microspheres G were eluted after incubation with the antibody IgG solution, the antibody IgG was captured and separated from the antibody solution, eluted and released into the eluate, and the corresponding purified antibody-containing eluate was subjected to SDS-PAGE test. gone. where M corresponds to the Marker molecular weight mark. Fig. 3 shows the RFU value measurement results of biomagnetic microsphere H (magnetic beads with anti-EGFP nano-antibodies) binding to eGFP protein. Biomagnetic Microsphere H and eGFP protein IVTT supernatant were incubated to bind the eGFP protein. Here, Total corresponds to IVTT supernatant not treated with biomagnetic microspheres and Flow-through corresponds to flow-through incubated once. Fig. 3 shows experimental results of separation and purification of eGFP protein using biomagnetic microsphere H (antiEGFP magnetic beads). Biomagnetic Microsphere H is eluted after incubation with eGFP protein solution, eGFP protein is captured and separated from the stock solution, eluted and released into the eluate, SDS-PAGE study of the eluate containing the corresponding purified eGFP protein. This is the result. where M corresponds to the Marker molecular weight mark.

The following will further describe the present invention with reference to specific embodiments and examples, while referring to the drawings. It should be understood that these examples are only illustrative of the invention and do not limit the scope of the invention. Experimental methods that do not specify specific conditions in the following examples are preferentially based on the conditions guided according to the above specific embodiments, followed by conventional conditions, such as "Sambrook et al., Molecular Cloning: Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free protein synthesis experiment manual", "Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis [methods] and .2008” and the conditions suggested by the manufacturer.

Unless otherwise stated, percentages and parts used herein refer to weight percentages and parts by weight.

Unless otherwise stated, all materials and reagents used in the examples of the present invention are commercially available products.

All temperature units in the present invention are degrees Celsius (° C.) unless otherwise specified.

Nucleotide and/or Amino Acid Sequence Listing SEQ ID No: 1, nucleotide sequence of protein A, 873 bases in length.
SEQ ID No: 2, nucleotide sequence of tamavidin2, 423 bases in length.
SEQ ID No: 3, nucleotide sequence of mEGFP, length is 714 bases.
SEQ ID No: 4, nucleotide sequence of protein G antibody binding site, 585 bases in length.
SEQ ID No: 5, amino acid sequence of anti-eGFP antibody, 117 amino acids in length.
SEQ ID No: 6, nucleotide sequence of mScarlet, 693 bases in length.

Nouns and Terms The following is an analysis or explanation of the meaning of "nouns", "terms" partly related to which the present invention employs, in order to better understand the present invention. A corresponding analysis or explanation applies to the full text of the invention, applies below, and applies above. When referring to cited documents in the present invention, the definitions of related terms, nouns, and phrases in the cited documents are also cited. If the definition in the cited document conflicts with the definition in the present invention, it shall not affect the cited component, substance, composition, material, system, formulation, type, method, equipment, etc. according to the content specified in

Magnetic beads: Ferromagnetic or possibly ferromagnetizable microspheres with a small particle size can also be described as magnetic beads, preferably with a diameter size of 0.1 μm to 1000 μm. Examples of magnetic beads of the present invention include magnetic microspheres A, magnetic microspheres B, magnetic microspheres C, biomagnetic microspheres D (biotin magnetic beads), biomagnetic microspheres F (protein A magnetic beads), biomagnetic microspheres Including but not limited to Sphere G (protein G magnetic beads), biomagnetic microsphere H (magnetic beads with antiEGFP nanoantibody), biomagnetic microsphere K (antibody magnetic beads).

Magnetic microsphere body: Magnetic beads with modified sites (magnetic microspheres with sites that can bind). For example, silica-coated magnetic material particles, more specifically aminated silica-coated magnetic material particles.

Magnetic microsphere A: Amino group-modified magnetic microsphere.

Magnetic microsphere B: A magnetic microsphere having a carbon-carbon double bond.

Magnetic Microsphere C: Acrylic polymer-modified magnetic microsphere.

Biotin Beads: Magnetic beads to which biotin or biotin analogues are bound and which are capable of binding specifically to avidin-type tagged substances. Its advantage is that the target protein can be labeled with avidin or a protein mutant of avidin and then integratedly expressed in the form of a fusion protein, which is convenient to use. Also called biotin microspheres. The biotin or biotin analogue herein can be a purification medium and can also be a linking element.

Biomagnetic Microspheres D: Magnetic microspheres bound with biotin or biotin analogues, biotin magnetic beads. Biotin herein can be a purification medium and can also be a linking element.

Avidin Beads: Magnetic beads to which avidin or an avidin analogue is bound. It can specifically bind to biotin-tagged substances. Also called avidin magnetic microspheres.

Biomagnetic microspheres F: Magnetic microspheres bound with protein A, protein A magnetic beads. A biotin-modified biomagnetic microsphere D and an avidin-protein A covalent complex E are bound to obtain.

Affinity protein magnetic beads: Magnetic microspheres bound with affinity proteins, which can be used for the separation and purification of antibody-like substances. Also called affinity protein microspheres.

Biomagnetic Microspheres G: Magnetic microspheres bound with Protein G, Protein G magnetic beads. For example, biotin-modified biomagnetic microspheres D and avidin-protein G covalent complexes are bound together.

Biomagnetic Microsphere K: Magnetic beads with attached antibody-type tags. It can be used for separation and purification of target substances that can specifically bind thereto. Also called antibody magnetic microspheres or antibody magnetic beads.

Nano-antibody magnetic beads: magnetic beads with attached nano-antibodies. It can be used for separation and purification of target substances that can specifically bind thereto. Also called nano-antibody magnetic microspheres.

Biomagnetic microspheres H: nanoantibody magnetic beads, magnetic microspheres bound with nanoantibody antiEGFP (antiEGFP magnetic beads). Avidin-antiEGFP covalent complex is bound to obtain.

Polymer: in the present invention broadly includes oligomers and macromolecules, having at least 3 structural units or having a molecular weight of at least 500 Da (said molecular weight is defined by any suitable characterization scheme, e.g. number average molecular weight, weight average molecular weight, viscosity average molecular weight can be employed).

Polyolefin chain: refers to a polymer chain containing no heteroatoms covalently bonded only by carbon atoms. In the present invention, a polyolefin backbone in a comb-like structure, such as the linear backbone of an acrylic polymer, is primarily concerned.

Acrylic polymer: Refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure, the copolymerization form of the copolymer is not particularly limited, and a linear main chain and weighed side groups COO- and the linear backbone of the acrylic polymer may contain heteroatoms. Here, the carbon-carbon double bond may have other substituents such as methyl substituents (corresponding to —CH ₃ C(COO—)—C—) as long as they do not hinder the progress of the polymerization reaction. may Here, COO- may exist in the form of -COOH, may be in the form of a salt (eg sodium salt), or in the form of a formate (preferably a formate alkyl ester, such as methyl formate -COOCH ₃ , ethyl formate-COOCH ₂ CH ₃ , hydroxyethyl formate-COOCH ₂ CH ₂ OH), and the like. Specific structural forms of the -C(COO-)-C- unit structure include -CH(COOH)-CH ₂ -, -CH(COONa)-CH ₂ -, -MeC(COOH)-CH ₂ -, - MeC(COONa)-CH ₂ -, -CH(COOCH ₃ )-CH ₂ -, -CH(COOCH ₂ CH ₂ OH)-CH ₂ -, -MeC(COOCH ₃ )-CH ₂ -, -MeC(COOCH ₂ CH ₂ OH)—CH ₂ —, etc., or any combination thereof. Here, Me is a methyl group. The linear main chain of one polymer molecule may contain only one type of unit structure (corresponding to a homopolymer), or may contain two or more types of unit structures. (corresponding to copolymer).

Acrylic monomer molecule: A monomer molecule used for synthesizing the above acrylic polymer, having a basic structure of C(COO-)=C, such as CH(COOH)=CH ₂ and CH(COONa)=CH ₂ , _CH3C (COOH)= _{CH2 ,} CH3C(COONa)= _CH2 , CH _{( COOCH3} ) _{= CH2} , CH( _COOCH2CH2OH )= _CH2 , CH3C( _COOCH3 )= CH ₂ , CH ₃ C(COOCH ₂ CH ₂ OH)=CH ₂ and the like.

Branched Chain: In the context of the present invention, a chain connected to a branch point and having independent ends. Branched chain and side branched chain in the present invention have the same meaning and can be used interchangeably. In the present invention, a branched chain means a side chain or a side group bonded to a linear main chain of a polymer, and the length and size of the branched chain are not particularly limited. It may be a short branched chain or a long branched chain with a large number of atoms. The structure of the branched chain is not particularly limited, and may be linear or have a branched structure. The branched chain may contain other side chains or side groups. Structural characteristics such as the number, length, size and degree of rebranching of the branched chains are preferably adjusted so as not to form a network structure as much as possible and increase the retention rate without causing accumulation of the branched chains, In this case, the flexible swing of the linear main chain can be exhibited smoothly.

Branched backbone: A branched backbone is one in which backbone atoms are sequentially linked in a covalent or non-covalent manner, from the branched chain ends to the backbone of the polymer. Branched backbones link functional groups at the ends of the polymer to the main chain of the polymer. The intersection of the branched backbone and the main chain is the branch point from which the branch is drawn. For example, the branched backbone between the purification medium and the polymer linear backbone is the affinity protein in the purification medium. —CH ₂ CH ₂ CH ₂ —NH—) and carbonyl groups (residues obtained by amidation reaction of carboxyl groups) can be sequentially linked to the polyolefin main chain of the polymer.

Branched chain ends include all branched chain ends. For the linear main chain, the end fixed to the main body of the magnetic microsphere and the other end of the linear main chain are always connected to one branch point, so the scope of the "branched chain end" of the present invention widely included in Accordingly, the polymer attached to the outer surface of the magnetic microsphere body of the present invention has at least one branch point.

Functional groups of polymer branches: have reactive activity, or have reactive activity after being activated, and can directly generate a covalent reaction with reactive groups of other raw materials, or have been activated It means that a covalent bond reaction with a reactive group of another raw material occurs later, and further a covalent bond is generated to connect. A functional group on the polymer branch is a specific binding site in one preferred embodiment.

The direct ligation system is a ligation system in which direct interaction occurs without intervening a spacer atom. The mode of interaction includes, but is not limited to, covalent mode, non-covalent mode, or combinations thereof.

An indirect connection scheme is a connection scheme formed via at least one linking element, in this case involving at least one spacer atom. Said linking elements include, but are not limited to, linking peptides, affinity complex links, and the like.

"Anchor" mode, such as anchoring, anchoring to, being anchored to, being anchored to, refers to a covalent mode of attachment.

The "ligation" / "binding" method such as with, is ligated, ligates to, ligates to, binds, captures, captures, etc. is not particularly limited, and includes covalent binding methods, non-covalent binding methods including but not limited to methods.

Covalent bonding method: A method of directly bonding with a covalent bond. The covalent bonding method includes, but is not limited to, dynamic covalent bonding method, and the dynamic covalent bonding method is a direct bonding method of dynamic covalent bonding.

Covalent bond: In addition to general covalent bonds such as amide bonds and ester bonds, reversible dynamic covalent bonds are also included. The covalent bond includes dynamic covalent bond. Dynamic covalent bonds are chemical bonds that are reversible and include, but are not limited to, imide bonds, hydrazone bonds, disulfide bonds, or combinations thereof. A chemical engineer can understand its meaning.

Non-covalent bonding methods: including but not limited to supramolecular interactions such as coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions.

Supramolecular interactions: including, but not limited to, coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof.

A linking element, also called a linking group, means an element for linking two or more non-adjacent groups and includes at least one atom. The linking method between the linking element and the adjacent group is not particularly limited, and includes, but is not limited to, covalent bonding methods, non-covalent bonding methods, and the like. The internal connection method of the connecting member is not particularly limited, and includes, but is not limited to, methods such as covalent bonding method and non-covalent bonding method.

Covalent linking element: All spacer atoms from one end of the linking element to the other are linked in a covalent manner.

Specific binding site: In the present invention, the specific binding site is a group or structural site having a binding function on the polymer branch chain, and the group or structural site specifically recognizes a specific target substance, Having a binding function, the specific binding can be achieved by bonding actions such as coordination, complexation, electrostatic forces, van der Waals forces, hydrogen bonding, covalent bonding, or other interactions.

Covalent Complex: Compounds obtained by direct or indirect covalent linkage, also called covalent conjugates.

Avidin-Purification Medium Covalent Complex: A compound linked in a covalent fashion, with avidin on one end and purification medium on the other, both linked directly via a covalent bond or via a covalent linking element. indirectly linked.

Avidin-affinity protein covalent complex E: an avidin-purification medium covalent complex with an affinity protein as a purification medium, or referred to as avidin-affinity protein complex E, with compounds linked in a covalent manner. with avidin at one end and an affinity protein at the other end, both of which are directly linked by a covalent bond or indirectly linked via a covalent linking group. The covalent linkage schemes include, but are not limited to, covalent bonds, connecting peptides, and the like. For example, streptavidin-protein A complex, streptavidin-protein A fusion protein, streptavidin-enhanced green fluorescent protein-protein A fusion protein (Protein A-eGFP-Streptavidin), Protein A-eGFP-Tamvavidin2, Protein G-eGFP-avidin fusion protein, Protein G-eGFP-Tamvavidin2, and the like.

Affinity complex: A non-covalent complex formed by two or more molecules with very strong affinity due to specific binding action, e.g. biotin (or biotin analogue) and avidin (or avidin (analogues). Methods for binding affinity complexes between biotin and avidin are well known to those skilled in the art.

A purified substrate, also called a target substance, is one that is separated from a mixed system. The purification substrate in the present invention is not particularly limited, but the purification substrate is preferably a proteinaceous substance (in this case, also referred to as a target protein).

The purification medium is capable of specifically binding the purification substrate, thereby capturing the purification substrate and further separating the purification substrate from the mixed system. Purification media attached to the branched ends of the polymers of the present invention are functional elements that have the ability to bind purification substrates. When the purification medium is covalently linked to adjacent groups, it is referred to as a functional group capable of binding the purification substrate.

Affinity protein: A protein that specifically binds to a target protein and has a relatively high affinity binding force. Examples include protein A, protein G, protein L, denatured protein A, denatured protein G, and denatured protein L. be done.

Protein A: Protein A, a 42 kDa surface protein, first found in the cell wall of Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topological structure, cell osmotic pressure and a binary system called ArlS-ArlR. It is used in biochemical research because of its ability to bind immunoglobulins. It can specifically bind to antibody Fc, is mainly used for antibody purification, and can be selected from any commercially available products. The so-called "Protein A", "SPA" and "Protein A" in the present invention are interchangeable.

Protein G: Protein G, an immunoglobulin binding protein, expressed in group C and group G streptococci, similar to protein A but with different binding specificities. It is a cell surface protein of 65 kDa (G148 protein G) and 58 kDa (C40 protein G) and is mainly used for antibody purification by specifically binding antibodies or certain functional proteins, selected from any commercial be able to.

Protein L: Protein L, limited to those that specifically bind to antibodies containing kappa (κ) light chains. In humans and mice, most antibody molecules contain a kappa light chain and a residual lambda light chain. It is mainly used for antibody purification and can be selected from any commercially available products.

Biotin: Biotin, capable of binding to avidin, has a strong binding force, and is excellent in specificity.

Avidin: avidin, which can bind to biotin, has strong binding force, and has excellent specificity, such as streptavidin (abbreviated as Streptavidin, SA), its analogues (for example, Tamvavidin2, abbreviated as Tam2), its denatured products, variants thereof, and the like.

A biotin analogue is a non-biotin molecule capable of forming a specific bond with avidin similar to "avidin-biotin", preferably a polypeptide or protein. ] series (e.g., Strep[R]II, Twin-Strep-tag[ R ], etc.), and analogous WN HPQFEK sequences (SEQ ID No: 10) . WNHPQFEK (SEQ ID No: 10) can be considered a variant sequence of WS HPQFEK (SEQ ID No: 9) .

An avidin analogue is a non-avidin molecule, preferably a polypeptide or protein, capable of forming a specific binding with avidin similar to "avidin-biotin". The avidin analogues include, but are not limited to, avidin derivatives, avidin homologues, avidin variants, and the like. Said avidin analogues are, for example, Tamavidin1, Tamavidin2, etc. (see literature: FEBS Journal, 2009, 276, 1383-1397).

Biotin-type tag: The biotin-type tag includes units of biotin, biotin analogues capable of binding to avidin, biotin analogues capable of binding to avidin analogues, and combinations thereof. A biotin-type tag can specifically bind avidin, an avidin analog, or a combination thereof. Therefore, a protein-based substance labeled with an avidin-type tag can be separated and purified, but is not limited to this.

Avidin-type tag: The avidin-type tag includes units of avidin, avidin analogues capable of binding to biotin, avidin analogues capable of binding to biotin analogues, and combinations thereof. Avidin-type tags can specifically bind biotin, biotin analogs, or combinations thereof. Therefore, a protein-based substance labeled with a biotin-type tag can be separated and purified, but it is not limited to this.

Polypeptide-type tags: A polypeptide-type tag of the present invention is a tag comprising a polypeptide tag or a derivative of a polypeptide tag. The polypeptide-type tag is a tag with a polypeptide structure composed of amino acids, and the amino acids may be natural amino acids or non-natural amino acids.

Protein-based tag: A protein-based tag of the invention is a tag comprising a protein tag or a derivative of a protein tag. The protein-type tag is a protein structure tag composed of amino acids, and the amino acids may be natural amino acids or non-natural amino acids.

Antibody-type tag: The antibody-type tag of the present invention is a tag containing an antibody-based substance, and can specifically bind to a target substance such as an antigen. Examples of the antibody-type tags further include antiEGFP nano-antibodies that can specifically bind to eGFP proteins.

Antigen-type tag: The antigen-type tag of the present invention is a tag containing an antigen-based substance and capable of specifically binding to an antibody-based substance.

Peptides are compounds in which two or more amino acids are linked by peptide bonds. In the present invention, peptide and peptide segment have equivalent meaning and can be used interchangeably.

Polypeptides are peptides composed of 10-50 amino acids.

Proteins are peptides composed of 50 or more amino acids. Fusion proteins are also proteins.

Derivatives of Polypeptides, Derivatives of Proteins: It is to be understood that any polypeptide or protein according to the invention also includes derivatives thereof, unless otherwise specified (eg, specifying a specific sequence). The polypeptide derivatives and protein derivatives include at least C-terminal containing tags, N-terminal containing tags, C-terminal and N-terminal containing tags. Here, the C-end refers to the COOH-end and the N-end refers to the _NH2 -end, and those skilled in the art will understand the meaning. Said tag may be a polypeptide tag or a protein tag. Some examples of tags include histidine tags (generally containing at least 5 histidine residues, such as 6xHis, HHHHHH, and also, for example, 8xHis tags), Glu-Glu, c-myc epitopes. (EQKLISEEDL , SEQ ID No: 25 ), FLAG [registered trademark] tag (DYKDDDDK , SEQ ID No: 19 ), protein C (EDQVDPRLIDGK , SEQ ID No: 26 ), Tag-100 (EETARFQPGYRS , SEQ ID No: 27 ) , V5 epitope label (V5 epitope, GKPIPNPLLGLDST , SEQ ID No: 28 ), VSV-G (YTDIEMNRLGK , SEQ ID No: 29 ), Xpress (DLYDDDDK, SEQ ID No: 30 ), hemagglutinin, YPYDVQ : 24 ), β-galactosidase, thioredoxin, histidine site thioredoxin (His-patch thioredoxin), IgG-binding domain (IgG-binding domain), intein-chitin binding domain , T7 gene 10, glutathione-S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), and the like.

A proteinaceous substance in the present invention is broadly defined as a substance containing a polypeptide or a protein fragment. For example, polypeptide derivatives, protein derivatives, glycoproteins, etc. are also included within the scope of proteinaceous substances.

Antibodies, Antigens: Antibodies and antigens of the present invention should be understood to include domains, subunits, fragments, single chains, single chain fragments and variants thereof, unless otherwise specified. For example, reference to an "antibody" includes fragments thereof, heavy chains, heavy chains lacking light chains (eg, nanobodies), complementarity determining regions (CDRs), etc., unless otherwise specified. For example, "antigen" further includes epitopes and epitope peptides, unless otherwise specified.

In the present invention, antibody-based substances refer to antibodies, antibody fragments, antibody single chains, single chain fragments, antibody fusion proteins, antibody fragments, as long as they can generate an antibody-antigen specific binding action. including, but not limited to, fusion proteins and the like, and derivatives and variants thereof.

In the present invention, antigen-based substances include, but are not limited to, antigens known to those skilled in the art, and substances that exert antigenic functions and specifically bind to antibody-based substances.

Anti-protein antibody: refers to an antibody that can specifically bind to a protein.

Anti-fluorescent protein nanobody: Refers to a nanobody that can specifically bind to a fluorescent protein.

Homology, unless otherwise stated, refers to having at least 50% homology, preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology. homologous, more preferably at least 80% homologous, more preferably at least 85% homologous, more preferably at least 90% homologous, and also for example at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. References include, for example, homologous sequences of the Ω sequences referred to in the description of the present invention. Homology here means sequence similarity, and may be numerically equivalent to identity.

A homologue refers to a substance with a homologous sequence, also called a homologue.

A "variant", variant, refers to a substance that has a different structure (including but not limited to making minor mutations) but retains or can essentially retain its original function or performance. Said variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. The manner in which related variants are obtained includes, but is not limited to, recombinations, deletions or deletions, insertions, shifts, substitutions, etc. of structural units. Said variants include, but are not limited to, modified products, genetically modified products, fusion products, and the like. Methods of genetic modification to obtain genetically modified products include, but are not limited to, genetic recombination (corresponding to genetically modified products), gene deletions or deletions, insertions, shifts, base substitutions, and the like. Gene mutation products, also called genetic variants, belong to one type of genetic modification products. One preferred form of said variant is a homologue.

Modified Products: Including but not limited to chemical modification products, amino acid modifications, polypeptide modifications, protein modifications and the like. The chemically modified products are products modified using chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry. Modification methods include ionization, salification, desalination, complexation, decomplexation, chelation, dechelation, addition reaction, substitution reaction, removal reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification. Specific examples include modification methods such as oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfurization.

Unless otherwise specified in the present invention, a "mutant" refers to a mutation product that retains or can essentially retain its original function or performance, and the number of mutation sites is not particularly limited. Said variants include, but are not limited to, genetic variants, polypeptide variants, protein variants. A mutant is a type of variant. The manner in which related variants are obtained includes, but is not limited to, recombination, deletion or deletion, insertion, shift, substitution, etc. of structural units. The structural unit of genes is the base, and that of polypeptides and proteins is the amino acid. Types of genetic mutations include, but are not limited to, gene deletions or deletions, insertions, shift coding, base substitutions, and the like.

"Modified" products include, but are not limited to, derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, which can retain their original function or performance, and which Functionality or performance may be optimized or changed.

Eluate (eg target protein): to elute the target protein, after elution the target protein is present in the eluate.

Wash solution (eg target protein): to elute the impurity protein, which after elution is carried away by the wash solution.

Avidity: Binding capacity, eg, binding capacity of a magnetic microsphere to a protein.

Affinity potency: Substrate concentration when magnetic microspheres bind only 50% of the substrate using substrate solutions with different concentration gradients.

IVTT: In vitro transcription and translation, an in vitro transcription and translation system, ie a cell-free protein synthesis system. A cell-free protein synthesis system uses an exogenous target mRNA or DNA as a template for protein synthesis, and adds substances such as substrates and transcription- and translation-related protein factors required for protein synthesis through artificial control to achieve target protein synthesis. can be done. The cell-free protein synthesis system of the present invention is not particularly limited, and any cell-free protein synthesis system based on yeast cell extract, E. coli cell extract, mammalian cell extract, plant cell extract, insect cell extract, or Any combination may be used.

In the present invention, "translation-related enzymes", translation-related enzymes (TRENs), means enzymatic substances necessary for the process of synthesizing a protein product from a nucleic acid template, and is not limited to enzymes necessary for the translation process. do not have. Nucleic acid template: Also called genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates and combinations thereof.

Flow-through: Clear fluid collected after incubating a system containing magnetic beads and target proteins, of which it contains residual target proteins not captured by the magnetic beads. For example, in Example 2, a solution containing a complex of an avidin-affinity protein is added to an affinity column, and is the solution that exits after passing through the column. The 1st, 2nd and 3rd run out solutions are shown.

RFU is Relative Fluorescence Units.

eGFP: enhanced green fluorescence protein. In the present invention, the eGFP broadly includes wild type and variants thereof, including but not limited to wild type and variants thereof.

mEGFP: A206K variant of eGFP.

"Optionally" indicates that the technical solution of the present invention can be implemented as a selection criterion, and may or may not be present. In the present invention, "arbitrary method" indicates that the present invention can be implemented by applying the technical idea of the present invention.

In the present invention, preferred embodiments such as "preferred (for example, prefer, preferred, preferred, preferred, etc.)", "preferably", "more preferred", "further preferred", and "most preferred" refer to the scope of the invention and It does not limit the scope of protection in any way, and does not limit the scope or embodiments of the present invention, but merely exemplifies some embodiments.

In the description of the present invention, "preferred one", "preferred form", "preferred embodiment", "preferred example", "preferred example", "in a preferred mode of implementation", "in some preferred examples ", "in some preferred forms", "preferred", "preferred", "preferred", "more preferred", "more preferred", "further preferred", "most preferred", etc. and schematic recitations such as "one embodiment", "one form", "illustration", "example", "example", "example", "for example", "example", "such as" The scheme likewise does not in any way constitute a limitation on the scope and protection of the invention, and the specific features described in each form are included in at least one specific embodiment of the invention. In the present invention, the specific features described in each form can be combined in any suitable form in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred form can be combined in any suitable form.

In the present invention, "any combination thereof" means numerically "greater than 1", range-wise "any one may be selected, or a group consisting of at least two You may."

In the present invention, the description of "one or more" such as "one or more", "one or more" means "at least one", "at least one", "a combination thereof", "or a combination thereof", " and any combination thereof", "or any combination thereof", "and any combination thereof", etc., have the same meaning and can be used interchangeably, and that the quantity is equal to "one" or "one or more" indicates

In the present invention, "or/and" is employed, and "and/or" represents "arbitrarily one or a combination thereof" and indicates at least one of them.

The conventional technical means described in the manner of "usually", "usually", "general", "always", "usually", etc. described in the present invention are all cited as reference for the content of the present invention, Unless otherwise stated, it can be regarded as a preferred form of some technical features of the present invention, and it should be noted that it does not constitute any limitation on the scope and protection scope of the invention in any way.

All documents mentioned in the present invention and documents directly or indirectly cited from those documents are incorporated by reference in this application, and each document appears to be individually cited by reference.

It should be understood that within the scope of the present invention, each of the above technical features of the present invention and each of the technical features specifically described below (including but not limited to the examples) can only be combined with each other to form new or preferred technical solutions and implement the present invention. Due to space limitations, I will not repeat all of them.

1. A first aspect of the present invention provides biomagnetic microspheres, said biomagnetic microspheres comprising a magnetic microsphere body, the outer surface of said magnetic microsphere body having a linear backbone and branched chains. one end of the linear main chain is fixed to the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the polymer of the magnetic microsphere is ligated to the branched end of biotin or a biotin analogue.

The biomagnetic microspheres of the first aspect of the invention are also called biotin magnetic microspheres or biotin magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG.

Said biotin or biotin analogue can not only serve as a purification medium, but can also be linked as a linking element to still other types of purification media.

Most commercial microspheres employ agarose-based materials, as compared to gel-based porous materials commonly used today, such as agarose-based materials. Porous materials have a rich pore structure, which provides a large specific surface area and high binding capacity for purification matrices, but correspondingly, protein molecules are porous when adsorbing or eluting proteins. Requires additional entry or exit through complex pore passages inside the material, which is more time consuming and prone to retention. In contrast, the binding site for target protein capture provided by the present invention utilizes only the outer surface space of the biomagnetic microspheres, and does not need to pass through complicated network passages during adsorption and elution, and the elution solution can be released directly into the water, thereby greatly reducing the elution time, improving the elution efficiency, reducing the retention rate and improving the purification yield.

1.1. Magnetic Microsphere Bodies In the present invention, the volume of the magnetic microsphere bodies can be of any viable particle size.

A relatively small particle size helps ensure that the magnetic microspheres are suspended in a mixed system and have more intimate contact with the protein product, improving the capture efficiency and binding rate of the protein product. In some preferred embodiments, the diameter size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0 .5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm , 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm , 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 500 μm, 500 μm , 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm, any one particle size scale or any two particle size scales (with deviations of ±25%, ±20%, ±15% , may be ±10%). Unless otherwise stated, the diameter size refers to the average size.

The volume of the magnetic microsphere body can be of any viable particle size.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.1-10 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.2-6 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.4-5 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.5-3 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.2-1 μm.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 0.5-1 μm.

In some preferred forms, said magnetic microsphere bodies have an average diameter of and the approximate number may be ±25%, ±20%, ±15%, ±10%.

In some preferred forms, the diameter of said magnetic microsphere body is selected from 1 μm to 1 mm.

In some preferred forms, the diameter of the magnetic microsphere body is e.g. %.

Different magnetic materials can provide different types of activation sites, can produce different modes of binding with the purification medium, and have different abilities to disperse and sediment with a magnet, and can also vary with the purification substrate type. Selectivity can be generated.

Magnetic microsphere bodies, and magnetic microspheres comprising magnetic microsphere bodies, can be rapidly positioned, guided, and separated under the action of an applied magnetic field, while multiple magnetic microsphere surfaces are formed on the magnetic microsphere surfaces by methods such as surface modification or chemical polymerization. can be provided with active functional groups such as hydroxyl, carboxyl, aldehyde, and amino groups, and the magnetic microspheres can bind biologically active substances such as antibodies and DNA by covalent or non-covalent methods. be able to.

In some preferred forms, the magnetic microsphere body is a magnetic material coated with _SiO2 . Here, the _SiO2 coating layer may contain a silane coupling agent having an active site on itself.

In some preferred forms, the magnetic material is an iron compound (e.g., iron oxide), iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide, manganese alloy, zinc oxide, gadolinium oxide. , chromium oxide, and combinations thereof.

In some preferred forms, the iron oxide is, for example, magnetite (Fe ₃ O ₄ ), maghemite (γ-Fe ₂ O ₃ ) or a combination of the two oxides, preferably triiron tetroxide. be.

In some preferred forms, the magnetic material is Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , AlNi(Co), FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo , MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), _FeSiAl , _BaO.6Fe2O3 , _SrO.6Fe2O3 , _{PbO.6Fe2O3 , GdO} and their Selected from a combination. Here, Re is rhenium, which is a rare earth element.

1.2. A polymeric structure that provides a large amount of branched chain ends The outer surface of the magnetic microsphere body has at least one polymer with a linear backbone and branched chains, and one end of the linear backbone is the magnetic microsphere body The other end of the polymer is free from the outer surface of the magnetic microsphere body.

The term "fixed to" refers to being "fixed to" the outer surface of the magnetic microsphere body in a covalent manner.

In some preferred forms, the polymer is covalently attached to the outer surface of the magnetic microsphere body either directly or indirectly via a linking element.

Said polymer has a linear backbone, in which case the polymer not only has the high flexibility of the linear backbone, but also has the advantage of high-fold amplification of the number of branched chains, enabling high-rate and high-throughput binding. , high-efficiency, high-ratio (high-yield) separations can be better achieved.

For the magnetic microspheres of the present invention, one end of the polymer is covalently attached to the outer surface of the magnetic microsphere body, and the remaining ends containing all branched chains and all functional groups are both dissolved in a solution, and the magnetic microsphere body is The molecular chains can be sufficiently stretched and oscillated, and the molecular chains can be sufficiently contacted with other molecules in the solution, further enhancing the capture of the target protein. . When the target protein is eluted from the magnetic microspheres, it directly escapes from the confinement of the magnetic microspheres and enters directly into the eluate. Polymers covalently anchored at one end by such linear backbones (some In preferred embodiments, a single single polymer linear backbone is covalently anchored, and in some other preferred embodiments, two or three linear backbones are covalently drawn from the anchored ends of the backbone. ) can effectively reduce the stacking of molecular chains, enhance the extension and rocking of molecular chains in solution, enhance the capture of target proteins, and reduce the retention rate and retention time of target proteins during elution. do.

1.2.1. Polymer Backbone of Biomagnetic Microspheres Provided by the Present Invention In some preferred forms, the linear backbone is a polyolefin backbone or an acrylic polymer backbone.

In some other preferred forms, the linear backbone of the polymer is an acrylic polymer backbone. It may be a polyolefin backbone (the linear backbone is only carbon atoms) or it may contain heteroatoms in the linear backbone (the heteroatoms are non-carbon atoms).

In some preferred forms, the polymer backbone is a polyolefin backbone. The monomer unit of the acrylic polymer is an acrylic monomer molecule such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof. The acrylic polymer may be obtained by polymerizing any of the above monomers, or may be obtained by copolymerizing an appropriate combination of the above monomers.

In some preferred forms, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin main chain is a main chain provided to a polymer of any one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, and methacrylate monomer. , or the backbone provided to the polymerization product of a combination thereof (the backbone provided to the copolymer thereof), or the backbone of the copolymer formed when the above monomers participate in the polymerization . Polymerization products obtained by combining the above monomers include, for example, acrylic acid-acrylic acid ester copolymer, methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate. A copolymer etc. are mentioned. Examples of copolymers formed through polymerization of the above monomers include maleic anhydride-acrylic acid copolymers.

In some preferred forms, the linear backbone is a polyolefin backbone and is provided from an acrylic polymer backbone.

In some preferred forms, the linear backbone is an acrylic polymer backbone.

In some other preferred forms, the polymer backbone is an acrylic polymer backbone. It may be a polyolefin backbone (the backbone is only carbon atoms) and may contain heteroatoms (heteroatoms: non-carbon atoms) in the backbone.

In some other preferred forms, the polymer backbone is a block copolymer backbone comprising polyolefin blocks, such as polyethylene glycol-b-polyacrylic acid copolymer (which belongs to the range of acrylic copolymers). ). It is preferred that the flexible rocking of the linear backbone is smoothly exerted without causing branch chain build-up and increasing the residence time or/and rate.

In some other preferred forms, the polymer backbone is a polycondensed backbone. The polycondensation type main chain means a linear main chain that can be formed by a polycondensation reaction between monomer molecules or oligomers. The polycondensation-type main chain may be homotype or copolymer type. For example, polypeptide chains, polyamino acid chains, and the like. Specific examples include ε-polylysine chains, α-polylysine chains, γ-polyglutamic acid chains, polyaspartic acid chains, and aspartic acid/glutamic acid copolymers.

The number of linear backbones to which one binding site on the outer surface of the magnetic microsphere body can be covalently bound may be one or more.

In some preferred forms, one binding site on the outer surface of the magnetic microsphere body can pull out only one linear backbone, providing a large working space for the linear backbone.

In some other preferred forms, one binding site on the outer surface of the magnetic microsphere body pulls out only two linear backbones, providing as much activity space for the linear backbones as possible.

The backbone of the polymer is covalently attached at one end to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), and the remaining ends containing all branches and all functional groups are both dissolved in solution and magnetically Distributed in the outer space of the beads, the molecular chains can be sufficiently stretched and oscillated, which allows the molecular chains to make sufficient contact with other molecules in the solution, further enhancing the capture to the target protein. be able to. When the target protein is eluted from the magnetic beads, it is directly released from the confinement of the magnetic beads and directly into the eluate. Polymers covalently anchored by one end of the linear backbones provided herein (most preferably single A single polymer linear backbone is covalently anchored, and preferably two or three linear backbones are covalently drawn to the anchored end of the backbone) effect stacking of the molecular chains. can be reduced exponentially, enhancing the stretching and rocking of the molecular chains in solution, enhancing capture of the target protein, and reducing the retention rate and residence time of the target protein during elution.

1.2.2. Polymer branched chains of the biomagnetic microspheres provided by the present invention The number of said branched chains is the size of the magnetic microsphere body, the backbone structure type of the polymer, the chain density on the outer surface of the magnetic microsphere body of the polymer (particularly the branched chains). density).

The number of polymer branches is multiple and at least three. The number of side branches is related to the size of the magnetic microsphere, the length of the polymer backbone, the linear density of the side branches along the polymer backbone, and the polymer to factors such as chain density at the outer surface of the magnetic microsphere. Related. The number of polymer branched chains can be controlled by controlling the charging ratio of raw materials.

The branched polymer has at least three branches.

Each branched chain end may or may not be independently bound to a purification medium.

When a purification medium is bound to the branched chain ends, each branched chain end may be independently bound directly to the purification medium, or may be indirectly bound via a linking element. good.

When purification media are attached to the ends of branched chains, the number of purification media may be one or more.

In some preferred forms, one molecule of said branched polymer is associated with at least three purification media.

1.3. Binding Mode of Biotin or Biotin Analog The mode in which the biotin or biotin analogue is linked to the branched end of the polymer is not particularly limited.

The manner in which the biotin or biotin analog is linked to the branched ends of the polymer includes, but is not limited to, covalent bonding, supramolecular interactions, or combinations thereof.

In some preferred forms, said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof. be.

In some preferred forms, the branched chains of the polymer are covalently attached to biotin or biotin analogues by functional group-based covalent bonds, covalently attaching biotin or biotin analogues to the ends of the polymer branches. It can be obtained by covalent reaction of biotin or biotin analogues with functional groups contained in branched chains of polymer molecules on the outer surface of biomagnetic microspheres. Here, one of the preferred embodiments of said functional group is a specific binding site (see "Nouns and Terms" section of specific embodiments for definition).

The covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group. Preferably, said functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. One of the preferred salt forms of the carboxyl group is a sodium salt form, such as COONa, and the preferred salt form of the amino group may be an inorganic salt form or an organic salt form. may also include, but are not limited to, hydrochloride, hydrofluoride, and like forms. The "combination of functional groups" refers to all branches of all polymer molecules on the outer surface of one magnetic microsphere, which can participate in the formation of covalent bonds based on different functional groups. Taking biotin as an example, all biotin molecules on the outer surface of one biotin magnetic microsphere can be covalently bound with different functional groups, but one biotin molecule can only be bound with one functional group. .

2. A second aspect of the present invention is based on said biomagnetic microspheres provided in the first aspect of the present invention, wherein said biotin or biotin analogue is further linked to a purification medium as a linking element. provide a sphere. That is, the branched ends of the polymer are linked to the purification medium via a linking element, the linking element comprising biotin or a biotin analogue.

purification element
A purification medium is a functional requirement that specifically captures a target substance from a mixed system, ie, a specific binding can occur between the purification medium and target substance molecules to be separated and purified. The captured target substance molecules are eluted and released under appropriate conditions, thereby achieving the purpose of separation and purification.

When the target substance is a protein substance, the purification medium can form a specific binding action with the target protein itself or with a purification tag carried by the target protein. Therefore, any substance that can be used for a target protein purification tag can be a selectable format for a purification medium, and a peptide or protein used as a purification medium can be a selectable format for a purification tag for a target protein. can do.

2.1. Types of Purification Media The purification media may contain, but are not limited to, avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In some preferred forms, the purification medium comprises avidin, an avidin analogue capable of binding to biotin or an analogue thereof, biotin, a biotin analogue capable of binding to avidin or an analogue thereof, an affinity protein, an antibody, an antigen, a DNA , or a combination thereof.

In some preferred forms, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin. do.

In some preferred forms, the avidin is streptavidin, denatured streptavidin, streptavidin analogs, or a combination thereof.

Said avidin analogues are, for example, tamavidin 1, tamavidin 2 and the like. Ｔａｍａｖｉｄｉｎ１及びＴａｍａｖｉｄｉｎ２は、Ｙａｍａｍｏｔｏらが２００９年に発見したビオチン結合能力を有するタンパク質であり（Ｔａｋａｋｕｒａ Ｙ ｅｔ ａｌ．Ｔａｍａｖｉｄｉｎｓ：Ｎｏｖｅｌ ａｖｉｄｉｎ－ｌｉｋｅ ｂｉｏｔｉｎ－ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎｓ ｆｒｏｍ ｔｈｅ Ｔａｍｏｇｉｔａｋｅ ｍｕｓｈｒｏｏｍ［Ｊ］．ＦＥＢＳ Ｊｏｕｒｎａｌ，２００９， 276, 1383-1397), they have a strong affinity for biotin similar to streptavidin. The thermostability of Tamavidin2 is superior to that of streptavidin, and its amino acid sequence can be retrieved from relevant databases, such as UniProt B9A0T7, and can also be codon-transformed and optimized by an optimization program to obtain the DNA sequence.

Said biotin analogues are, for example, the WSHPQFEK sequence (SEQ ID No: 9) or its variant sequence, the WRHPQFGG sequence (SEQ ID No: 7) or its variant sequence, and the like.

In some preferred forms, the purification medium is a polypeptide tag, protein tag, or a combination thereof.

In some preferred forms, the purification medium is an affinity protein.

Examples of said affinity proteins include, but are not limited to, protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, and the like.

The definitions of the antibodies and antigens refer to the term part, domain, subunit, fragment, heavy chain, light chain, single chain fragment (e.g. nanobody, light chain deleted heavy chain, heavy chain variable region, complementarity determining regions, etc.), epitopes, epitope peptides, variants of any of the foregoing, and the like.

In some preferred forms, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a tag comprising a WSHPQFEK (SEQ ID No: 9) sequence; A tag containing a variant sequence of WSHPQFEK (SEQ ID No: 9) , a tag containing a WRHPQFGG (SEQ ID No: 7) sequence, a tag containing a variant sequence of WRHPQFGG (SEQ ID No: 7) , RKAAVSHW (SEQ ID No: 8 ) ) , a tag comprising a variant sequence of RKAAVSHW (SEQ ID No: 8) , or a combination thereof or variants thereof.

In some preferred forms, said protein tag is any tag or variant thereof selected from an affinity protein, a SUMO tag, a GST tag, an MBP tag, or combinations thereof, more preferably said affinity protein is , protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

In some preferred embodiments, the antibody-based tag is an antibody, a fragment of an antibody, a single chain of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any, or A variant of either.

In some preferred forms, the antibody-based tag is an anti-protein antibody.
In some preferred forms, the antibody-based tag is an anti-fluorescent protein antibody.
In some preferred forms, the antibody-based tag is a nanobody.
In some preferred forms, the antibody-based tag is an anti-protein nanobody.
In some preferred forms, the antibody-based tag is an anti-fluorescent protein nanobody.
In some preferred forms, the antibody-based tag is an anti-green fluorescent protein or variant thereof antibody.
In some preferred forms, said antibody-based tag is an Fc fragment.

2.2. Supporting Method of Purification Medium The method in which the purification medium is linked to the biotin or biotin analogue is not particularly limited.

The manner in which the purification medium is linked to the biotin or biotin analog includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or combinations thereof.

In some preferred forms, said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap interactions, hydrophobic interactions, and combinations thereof. be.

In some preferred forms of the biomagnetic microspheres, the purification media are linked to the branched ends of the polymer by linking elements comprising affinity complexes.

In some preferred forms, the biotin or biotin analogue is bound to avidin or an avidin analogue by affinity complexing, and the purification medium directly or indirectly binds to the avidin or avidin analogue.

In some preferred forms, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction .

In some preferred forms, the selection criteria for the affinity conjugate are to provide a highly specific, high affinity, yet chemically conjugable site whereby the affinity conjugate is covalently attached to the polymer branch ends. or can be covalently attached to the outer surface of the magnetic microsphere body after being chemically modified, e.g. be. For example, combinations of substances from biotin or its analogues and avidin or its analogues, antigens and antibodies and the like.

If the loading scheme involves dynamic covalent bonding, supramolecular interactions (especially affinity complex interactions), it forms a reversible loading scheme, and purification media can be unloaded from branched chain ends under certain conditions. , further update or replace.

Renewal of purification media corresponds to regeneration of magnetic microspheres, and the type of purification media before and after renewal is the same.

The exchange of purification media corresponds to changing the magnetic microspheres, and the types of purification media before and after the exchange are different.

In some preferred forms, the purification medium is avidin and further comprises biotin that binds to the avidin, wherein the biotin is a linking member and the binding of the affinity complex between biotin and avidin.

In some preferred forms, the purification medium is an affinity protein and further comprises avidin linked to the affinity protein and biotin linked to the avidin, wherein binding of the affinity complex between biotin and avidin form an action, with the affinity complex as the linking element.

In some preferred forms, biotin, avidin, and purification media are sequentially linked to the branched ends of the polymer of the biomagnetic microspheres. In a further preferred form, said purification medium is an antibody or antigen. Modes of linking the avidin with the purification medium include, but are not limited to, covalent bonds, non-covalent bonds, linking elements, or combinations thereof.

In some preferred forms, the purification medium comprises nucleic acids, oligonucleotides, peptide nucleic acids, nucleic acid aptamers, deoxyribonucleic acids, ribonucleic acids, leucine fasteners, helix turn helix motifs, zinc finger motifs, biotin, anti-biotin proteins, streptavidin, The biomagnetic microspheres are linked to the polymer branched ends of the biomagnetic microspheres by linking elements, including but not limited to combinations thereof, such as anti-hapten antibodies. Of course, said linking elements are selected from double-stranded nucleic acid structures, double helices, homohybrids or heterohybrids (DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA homohybrids or heterohybrids), or combinations thereof.

2.3. Mechanism of Action of the Purification Medium The force with which the purification medium captures target molecules in the reaction-purification mixture system is selected from, but not limited to, covalent bonds, supramolecular interactions, and combinations thereof.

In some preferred forms, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction .

In some preferred forms, the target substance has biotin-avidin binding capacity, Streg tag-avidin binding capacity, avidin-affinity protein binding capacity, histidine tag-metal ion affinity, antibody-antigen binding capacity, or combinations thereof. The biomagnetic microspheres are bound to the polymer branched chain ends by a force method. The Streg tag mainly includes, but is not limited to, a peptide tag that forms a specific binding activity with avidin or its analogue developed by IBA, and usually includes a WSHPQFEK sequence or a modified sequence thereof.

2.4. Regeneration and Reuse of Purification Media If the purification media is linked to the polymer branched ends of the biomagnetic microspheres of the invention by reversible methods such as affinity complexes, dynamic covalent bonding, etc., under appropriate conditions, the purification media can be combined with the polymer. The branched ends can be eluted and recombined with new purification media.

Affinity complex interactions are exemplified by affinity complex interactions between biotin and streptavidin.

The extremely strong affinity between biotin and streptavidin is the binding action of typical affinity complexes, stronger than common non-covalent binding actions and at the same time weaker than covalent binding actions, thereby allowing purification media to escape from magnetic beads. Not only can it bind tightly to the polymer branch ends on the surface, but also achieve synchronous release of the purification medium by eluting streptavidin from the specific binding site of biotin when the purification medium needs to be replaced. , further liberating the activation site of new avidin-purification media covalent complexes (e.g. streptavidin-tagged purification media), thereby enabling rapid recovery of magnetic bead purification performance and target substances (e.g. antibodies) significantly reduce the cost of separation and purification of The process of eluting the purification medium-modified biomagnetic microspheres and removing the avidin-purification medium covalent complexes, thereby recapturing the biotin or biotin analogue-modified biomagnetic microspheres is characterized by biotin magnetic properties. called microsphere regeneration. The renatured biotin-magnetic microspheres have released biotin active sites and can rebind avidin-purification medium covalent complexes, yielding again purification medium-modified biomagnetic microspheres ( (corresponding to regeneration of biomagnetic microspheres), fresh purification media can be provided, providing new target substance binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated and used, ie, the purification media can be exchanged and reused.

2.4. Purification substrate (preferably proteinaceous material)
The purification substrate of the present invention is a substance that is captured and separated by the magnetic microspheres of the present invention, and is particularly limited as long as it is a purification medium that can specifically bind to the magnetic microspheres of the present invention. isn't it.

When said purification substrate is a protein-based substance, the purification substrate is also called target protein.

2.4.1. Purification Tags in Target Proteins The target protein may not carry a purification tag, in which case the target protein itself should be captured in a purification medium in magnetic microspheres. For example, "target protein, purification medium" may be combinations such as "antibody, antigen", "antigen, antibody", "avidin or analogue thereof, biotin or analogue thereof".

In some preferred forms, the target protein is tagged with a purification tag, and the purification tag is capable of specifically binding to the purification medium. In one target protein molecule, the number of the purification tags is one, two or more, and when two or more purification tags are included, the types of purification tags are one, two or multiple. As long as the amino acid sequences of the tags are different, they are regarded as different types of tags.

The purification tag in the target protein includes a histidine tag, avidin, an avidin analogue, a Streg tag (a tag containing a WSHPQFEK sequence (SEQ ID No: 9) or a variant thereof), a WRHPQFGG sequence (SEQ ID No: 7) or a variant thereof. tags containing RKAAVSHW (SEQ ID No: 8) sequences or variants thereof, FLAG tags or variants thereof, C tags and variants thereof, Spot tags and variants thereof, GST tags and variants thereof, MBP tags and variants thereof, SUMO The above tag groups include, but are not limited to, tags and variants thereof, CBP tags and variants thereof, HA tags and variants thereof, Avi tags and variants thereof, affinity proteins, antibody-based tags, antigen-based tags, and combinations thereof. It can also be selected from the purification tags disclosed in US6103493B2, US10065996B2, US8735540B2, US20070275416A1, including but not limited to Streg tags and variants thereof.

The purification tag can be fused through the N-terminus or the C-terminus.

Said histidine tag generally comprises at least 5 histidine residues, such as a 5xHis tag, a 6xHis tag, an 8xHis tag, and the like.

The octapeptide WRHPQFGG (SEQ ID No: 7) can specifically bind core streptavidin.

The Streg tag is capable of forming a specific binding activity with avidin or an analogue thereof and includes WSHPQFEK (SEQ ID No: 9) or variants thereof in said Streg tag. For example, WSHPQFEK-(XaaYaaWaaZaa) _n -WSHPQFEK (SEQ ID No: 11) , where Xaa, Yaa, Waa, Zaa are each independently any amino acid and XaaYaaWaaZaa comprises at least one amino acid. and (XaaYaaWaaZaa) _n (SEQ ID No: 12) comprises at least 4 amino acids, wherein n is selected from 1 to 15 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15), (XaaYaaWaaZaa) _n (SEQ ID No: 12) include (G) ₈ , (G) ₁₂ , GAGA, (GAGA) ₂ , ( GAGA) ₃ , (GGGS) ₂ and (GGGS) ₃ . Streg tags include, for example, WSHPQFEK (SEQ ID No: 9) , WSHPQFEK-(GGGS) _n -WSHPQFEK (SEQ ID No: 13) , WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK (SEQ ID No: 14) , SA-WSHPQFEK-( GGGS) ₂ GGSA-WSHPQFEK (SEQ ID No: 15) , WSHPQFEK-GSGGG-WSHPQFEK-GL-WSHPQFEK (SEQ ID No: 16) , GGSA-WNHPQFEK-GGGSGSGGSA-WSHPQFEK-GS, GS-GS (SEQ ID No: 16) WSHPQFEK-GGGSGGGSGGSA-WSHPQFEK (SEQ ID No: 18) and the like.

The sequence of the FLAG tag is DYKDDDDK (SEQ ID No: 19) . Variant sequences of the FLAG tag include, for example, DYKDHD-G-DYKDHD-I-DYKDDDDK (SEQ ID No: 20) .

The sequence of the Spot tag is PDRVRAVSHWSS (SEQ ID No: 21) .

The C-tag contains an EPEA (SEQ ID No: 22) sequence.

The GST tag means a glutathione S-transferase tag.

The MBP tag is a maltose binding protein tag.

The SUMO tag is a known small ubiquitin-like modifier and one of the key members of the ubiquitin family of polypeptide chain superfamily. In primary structure, SUMO and ubiquitin share 18% homology, but their tertiary structures and their biological functions are quite similar.

The sequence of the CBP tag is KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID No: 23) .

The HA tag sequence is YPYDVPDYA (SEQ ID No: 24) .

The Avi-tag is a small tag composed of 15 known amino acid residues, which is specifically recognized by the biotin ligase BirA.

Antibody tags include complete antibody structures (whole antibodies), domains, subunits, fragments, heavy chains, light chains, single chain fragments (e.g. nanobodies, heavy chains with light chains deleted, heavy chain variable regions, complementarity determining regions, etc.).

Antigenic tags include, but are not limited to, complete antigen structures (complete antigens), domains, subunits, fragments, heavy chains, light chains, single chain fragments (eg, epitopes, etc.), and the like.

In some preferred forms, one purification tag is attached to the N-terminus or C-terminus of the target protein, or both ends are attached to purification tags.

Any of the various purification tags described in this section can be candidates for purification media in the magnetic microspheres of the present invention.

2.4.2. Type of Target Protein The target protein may be a natural protein or modified product thereof, or an artificial synthetic sequence. The origin of the natural protein is not particularly limited, and includes but is not limited to eukaryotic cells, prokaryotic cells, and pathogens, among which eukaryotic cell origins include mammalian cells, plant cells, yeast cells, insect cells, and lineages. Said mammalian cells, including but not limited to worm cells, and combinations thereof, may be derived from murine sources (including rats, mice, guinea pigs, golden gophers, hamsters, etc.), rabbit sources, monkey sources, human sources, Including, but not limited to, swine sources, sheep sources, bovine sources, dog sources, horse sources, and the like. Said pathogens include viruses, chlamydia, mycoplasma, and the like. Said viruses include HPV, HBV, TMV, coronaviruses, rotaviruses and the like.

The types of target proteins include polypeptides (the term "target protein" in the present invention broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagen, polypeptides. Including, but not limited to, amino acids, vaccines, etc., partial domains of any of the above proteins, subunits or fragments of any of the above proteins, and variants of any of the above proteins. The "subunit or fragment of any of the above proteins" includes subunits or fragments of "a partial domain of any of the above proteins." The "variants of any of the above proteins" include variants of "a partial domain of any of the above proteins, subunits or fragments of any of the above proteins". The "variants of any of the above proteins" include, but are not limited to, variants of any of the above proteins. In the present invention, situations and meanings of two or more "above" in succession in other positions are similarly explained.

The structure of the target protein may be a complete structure, or may be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, and the like. Incomplete antibody structures include, for example, _nanobodies (heavy chain antibodies with light chains deleted, VHH, retaining the full antigen-binding ability of heavy chain antibodies), heavy chain variable regions, complementarity determination region (CDR) and the like.

For example, target proteins that can be synthesized by the in vitro protein synthesis system according to the present invention are selected from, but limited to, any of the following proteins, fusion proteins in any combination, and compositions in any combination: not. namely, luciferase (eg, firefly luciferase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), ammonia acyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase. , mouse catalase), actin, antibodies, antibody variable regions (e.g. antibody single chain variable regions, scFV), antibody single chains and fragments thereof (e.g. antibody heavy chains, nanobodies, antibody light chains) , alpha-amylase, enterobacter A, hepatitis C virus E2 glycoprotein, insulin and its precursors, glucagon-like peptide (GLP-1), interferons (including interferon alpha, interferon beta, interferon gamma, such as interferon alpha A) ), interleukins (such as interleukin-1β, interleukin-2, interleukin-12), lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, A partial domain of any of the foregoing proteins, such as xylanase, β-galactosidase, LacZ, e.g. E. coli β-galactosidase, a subunit or fragment of any of the foregoing proteins, or a variant of any of the foregoing (defined above) As such, said variants include variants, eg, luciferase variants, eGFP variants, which variants may also be homologues). Examples of the ammonia acyl-tRNA synthetase include human lysine-tRNA synthetase and human leucine-tRNA synthetase. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN109423496A. A composition of any of the above combination modalities may comprise any of the proteins described above and may comprise a fusion protein of any of the above combination modalities.

In some preferred embodiments, a target protein with fluorescent properties, such as one of GFP, eGFP, mScarlet, etc., or analogues or variants thereof, is used to assess the protein synthesis capacity of the in vitro protein synthesis system. .

The application fields of said target protein include biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, biotechnology, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanotechnology, cosmetics, food, Including but not limited to fields such as food additives, nutrients, agriculture, feed, daily necessities, laundry, environment, chemical dyeing and fluorescent labeling.

2.4.3. Mixed Systems Containing Target Proteins The magnetic microspheres of the invention can be used to separate target proteins from their mixed systems. The target protein is not limited to one substance, but may be a combination of multiple substances, provided that the purpose of purification is to obtain this composition, or the form of this composition can meet the purification requirements. may

The mixed system containing the target protein is not particularly limited, and the purification medium for the magnetic microspheres of the present invention only needs to be able to specifically bind to the target protein. It is also necessary that there is no specific binding or non-specific binding activity of the compound with other substances.

In embodiments of the present invention, the mixed system containing the target protein may be of natural origin, artificially constructed or derived mixed system.

For example, a specific protein can be separated and purified from commercially available serum.

For example, the target protein can be separated from the post-reaction system of an in vitro protein synthesis system.

A specific embodiment of said in vitro protein synthesis system further includes, but is not limited to, the E. coli-based cell-free protein synthesis system described, for example, in WO2016005982A1. Other citations of the present invention are in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marcianas cited in the direct and indirect citations. and are not limited to, all of which are incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, in the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45", part "2.1 Systems and Advantage 2 on page 7 to 8" Any of the in vitro protein synthesis systems can be an in vitro protein synthesis system to practice the present invention, including, but not limited to, the in vitro cell-free protein synthesis systems described in the literature.例えば、（本発明と矛盾しない限り、以下の文献及びその参考文献をその全体及びすべての目的のために引用する）文献ＣＮ１０６９７８３４９Ａ、ＣＮ１０８５３５４８９Ａ、ＣＮ１０８６９０１３９Ａ、ＣＮ１０８９４９８０１Ａ、ＣＮ１０８６４２０７６Ａ、ＣＮ１０９０２２４７８Ａ、ＣＮ１０９４２３４９６Ａ、ＣＮ１０９４２３４９７Ａ、ＣＮ１０９４２３５０９Ａ、ＣＮ１０９８３７２９３Ａ 、ＣＮ１０９９７１７８３Ａ、ＣＮ１０９９８８８０１Ａ、ＣＮ１０９９７１７７５Ａ、ＣＮ１１００９３２８４Ａ、ＣＮ１１０４０８６３５Ａ、ＣＮ１１０４０８６３６Ａ、ＣＮ１１０５５１７４５Ａ、ＣＮ１１０５５１７００Ａ、ＣＮ１１０５５１７８５Ａ、ＣＮ１１０８１９６４７Ａ、ＣＮ１１０８４５６２２、ＣＮ１１０９３８６４９Ａ、ＣＮ１１０９６４７３６Ａ、ＣＮ１１１３７８７０６Ａ、ＣＮ１１１３７８７０７Ａ、ＣＮ１１１３７８７０８Ａ、ＣＮ１１１７１８４１９Ａ、ＣＮ１１１７４８５６９Ａ、ＣＮ２０１９１０７２９８８１３、ＣＮ２０１９１１２０６６１６３、ＣＮ２０１８１１２８６２０９３（ＣＮ１１１１１８０６５Ａ）、ＣＮ２０１９１１４１８１５１８、ＣＮ２０２０１００６９３８３３、 The cell-free protein synthesis system, DNA template construction and amplification method described in CN2020101796894, CN202010269333X, CN2020102693382 and references cited therein implement the in vitro protein synthesis system of the present invention, the DNA template construction and amplification method of the present invention. can be used to

The cells from which the cell extract of the in vitro protein synthesis system is derived are not particularly limited as long as they can express the target protein in vitro. Applied to in vitro protein synthesis system from prokaryotic cell extract, eukaryotic cell extract (preferably yeast cell extract, more preferably Kluyveromyces lactis) disclosed in the prior art Exogenous protein, or endogenous protein of prokaryotic, eukaryotic cell system (preferably yeast cell system, more preferably Kluyveromyces lactis system) applied for intracellular synthesis may be can also be synthesized using the in vitro protein synthesis system of the present invention, or attempted to be synthesized using the in vitro protein synthesis system provided by the present invention.

A preferred form of said in vitro protein synthesis system is the IVTT system. The liquid after the IVTT reaction (referred to as IVTT reaction liquid) contains, in addition to the expressed target protein, reaction raw materials remaining in the IVTT system, particularly various factors derived from cell extracts (e.g., ribosomes, tRNA, translation-related enzymes, etc.). , initiation factor, elongation factor, termination factor, etc.). The IVTT reaction solution can provide the target protein for binding to the magnetic beads, and can also provide a mixed system for verifying the separation effect of the target protein.

3. A third aspect of the present invention is based on said biomagnetic microspheres provided in the first aspect of the present invention, further comprising said biotin or biotin analogue as a linking member and further comprising avidin by affinity complex binding action. Or provide biomagnetic microspheres linked to an avidin analogue.

The biomagnetic microspheres of the third aspect of the invention are also called avidin magnetic microspheres or avidin magnetic beads.

Said avidin or avidin analogue can not only serve as a purification medium, but can also be linked with other types of purification media as a linking element. An affinity complex binding is now formed between said biotin or biotin analogue and said avidin or avidin analogue.

In some preferred forms, the biomagnetic microspheres provided in the first aspect of the invention further comprise avidin binding to the biotin. An affinity complex binding is now formed between biotin and avidin. That is, biotin is linked to the ends of the branched chains of the polymer of the magnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin.

In some preferred forms, the avidin is streptavidin, denatured streptavidin, streptavidin analogs, or a combination thereof.

4. A fourth aspect of the present invention is based on the biomagnetic microspheres provided in the third aspect of the present invention, further comprising an affinity protein linked to the avidin or avidin analogue. I will provide a. At this time, biotin or a biotin analogue and avidin or an avidin analogue form a binding action of an affinity complex between them as linking elements, and the affinity protein can not only serve as a purification medium, It can be a connecting element, preferably a purification medium.

The biomagnetic microspheres of the fourth aspect of the invention are also called affinity protein magnetic microspheres or affinity protein magnetic beads.

A typical structure of the biomagnetic microspheres is shown in FIG.

In some preferred forms, according to the biomagnetic microspheres provided in the second aspect of the invention, the purification medium is an affinity protein, and the biomagnetic microspheres are further linked to the affinity protein. and biotin bound to said binding force, wherein the purification medium is linked to the branched ends of said polymer by a linking element, said linking element formed by biotin and avidin. including affinity complexes.

In some preferred forms, the affinity protein is Protein A, Protein G, Protein L, or denatured proteins thereof. The corresponding biomagnetic microspheres are called Protein A magnetic microspheres or Protein A magnetic beads, Protein G magnetic microspheres or Protein G magnetic beads, Protein L magnetic microspheres or Protein L magnetic beads, etc. respectively.

The biomagnetic microspheres provided in the fourth aspect of the present invention are capable of strongly binding the affinity protein to the polymer branched ends on the outer surface of the magnetic bead, using the biotin-avidin-affinity protein linking method as an example. Alternatively, synchronous release of affinity proteins can be achieved by eluting avidin (e.g. streptavidin) from the specific binding sites of biotin when the affinity proteins need to be exchanged, and new avidin-affinity proteins The activated site of covalent complex E (eg, streptavidin-tagged affinity protein) can be reattached, resulting in rapid recovery of magnetic bead purification performance and greatly reduced antibody isolation and purification costs. The process of eluting the affinity protein-modified biomagnetic microspheres, removing the avidin-affinity protein covalent complex E, and thereby recapturing the biotin-modified biomagnetic microspheres, is the process of removing biotin-modified biomagnetic microspheres. called play. The regenerated biotin-magnetic microspheres have released biotin active sites and can rebind the avidin-affinity protein covalent complex E, again yielding affinity protein-modified biomagnetic microspheres F (biomagnetic (corresponding to regeneration of microspheres F) can be obtained to provide fresh affinity proteins and provide new antibody binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated and used, ie the affinity protein can be exchanged and reused.

5. A fifth aspect of the invention provides a method of making the biomagnetic microspheres provided in the first aspect of the invention.

5.1. Manufacture and Principles of the Biomagnetic Microspheres Provided in the First Aspect Provided in the first aspect of the invention are biotin magnetic microspheres modified with biotin or a biotin analogue.

Take for example the case where biotin is modified.
The biomagnetic microspheres provided in the first aspect provide magnetic beads (commercially available or homemade) coated with _SiO2 , activatingly modify the _SiO2 , and covalently bond a polymer to the _SiO2 (the polymer is Covalently bonded to SiO ₂ by one end of the linear backbone and a large amount of side branching distributed along the polymer backbone), biotin is covalently bonded to the branched chain ends of the polymer. By way of explanation, the above steps do not require complete isolation, allowing the integration of two or three steps into one step, e.g. activated silica-coated magnetic beads ( commercially available or homemade) can be provided directly. The step of activating and modifying SiO ₂ is, for example, step (1) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention. The step of covalently bonding the polymer to SiO ₂ is, for example, steps (2), (3) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention. The step of covalently bonding biotin to the ends of the polymer branched chains is, for example, step (4) of the method for producing biomagnetic microspheres according to the fifth to eighth aspects provided by the present invention.

The biomagnetic microspheres are produced by: ( ₁ ) providing or manufacturing a magnetic microsphere body and having reactive groups _R1 on the outer surface of the magnetic microsphere body; ( ₃ ) biotin or It can be prepared by linking biotin analogues.

Taking a magnetic material coated with _SiO2 as the main body of the magnetic microspheres, the biomagnetic microspheres are: (1) providing _SiO2 coated magnetic microspheres (commercially available or self _- made); (2) performing _{a polymerization reaction on the reactive group R 1 (} for example, with acrylic acid or sodium acrylate as a monomer molecule) to form a linear backbone and ( ₃ ) binding biotin or _a biotin analogue to the functional group F1 at the end of the branched chain. and can be manufactured by At this time, the polymer covalently attached to the magnetic microsphere body has a linear backbone, one end of the linear backbone is covalently attached to the reactive group _R1 , and a large amount of side chains along the polymer backbone. Branched chains are distributed.

5.2. Exemplary Example A typical method of manufacturing the biomagnetic microspheres (see FIG. 3) includes the following steps.

Step (1): providing a magnetic microsphere body, chemically modifying the magnetic microsphere body, introducing an amino group to the outer surface of the magnetic microsphere body, forming an amino group-modified magnetic microsphere A;

In some preferred forms, a coupling agent is utilized to chemically modify the magnetic microsphere body.

In some preferred forms, the silane coupling agent is an aminated silane coupling agent.

In some preferred embodiments, the magnetic microsphere body is a magnetic material coated with _SiO2 , and a silane coupling agent is used to chemically modify the magnetic microsphere body. The silane coupling agent is, in some preferred forms, an aminated silane coupling agent.

Step (2): Covalent bonding reaction between carboxyl group and amino group to covalently bond acrylic acid molecule to the outer surface of said magnetic microsphere A, introduce carbon-carbon double bond, carbon- Magnetic microspheres B containing carbon double bonds are formed.

Step (3): Using the polymerization reaction of carbon-carbon double bonds to polymerize acrylic monomer molecules (such as sodium acrylate), the resulting acrylic polymer is a branched polymer with a linear main chain and a branched chain containing functional groups F ₁ , the polymer is covalently bonded to the outer surface of magnetic microsphere B by one end of the linear backbone to form magnetic microsphere C modified with an acrylic polymer. This step can be performed without the addition of a cross-linking agent.

See the "Nouns and Terminology" section for definitions of the functional groups of the acrylic monomer molecules and polymer branches.

In some preferred forms, the functional group F ₁ is a carboxyl group, a hydroxyl group, an amino group, a mercapto group, a formate, ammonium, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or the functional group The above-mentioned "combination of functional groups" refers to functional groups contained in all branched chains of all polymers on the outer surface of one magnetic microsphere, and the types thereof are one or more. may be The meaning of "combination of functional groups" defined in the first aspect is consistent.

In some other preferred forms, the functional group is a specific binding site.

Step (4): Biotin or a biotin analogue is covalently attached to the branched end of the polymer by means of _a functional group F1 contained in the branched chain of the polymer, and biomagnetic microspheres (biotin magnetic microspheres). In the manufactured biomagnetic microspheres, an acrylic polymer (with a polyacrylic acid backbone) provides a large amount of sites to which biotin can be attached.

5.3. Specific Embodiments Specific embodiments for producing the biotin magnetic microspheres are as follows.

Specifically, taking as an example the acrylic polymer to provide a linear main chain and multiple branches, the present invention provides the following specific embodiments. With silica-coated triiron tetroxide magnetic beads as the main body of the biomagnetic microspheres, firstly, the coupling agent 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent, A silane coupling agent, more specifically an aminated silane coupling agent) is used to chemically modify the silica-coated triiron tetroxide magnetic beads to convert amino groups to the magnetic beads. It is introduced into the outer surface to complete the activation modification on _SiO2 to obtain amino group-modified magnetic microspheres A. Subsequently, the immobilized molecule (acrylic acid molecule providing one carbon-carbon double bond and one reactive group carboxyl group) is removed from the magnetic bead using covalent bonding reaction between the carboxyl group and the amino group. Covalently bound to the surface, thereby introducing carbon-carbon double bonds to the outer surface of the magnetic beads, obtaining magnetic microspheres B containing carbon-carbon double bonds. Subsequently, an acrylic monomer molecule (for example, sodium acrylate) is polymerized using the polymerization reaction of carbon-carbon double bonds, and the polymerization product is covalently bonded to the outer surface of the magnetic beads at the same time as the polymerization reaction is performed, The linking of the polymer to _SiO2 (covalent method) is completed to obtain acrylic polymer-modified magnetic microspheres C. The immobilized molecules here are acrylic acid molecules, one immobilized molecule pulls out only one polymer molecule and at the same time only pulls out one polymer linear backbone. Taking sodium acrylate as an example of the monomer molecule, the polymerization product is sodium polyacrylate, the main chain of which is a linear polyolefin main chain, and a plurality of side branched chains COONa are shared along the main chain. The functional group that is attached and included in the branched chain is also COONa. The polymerization reaction here does not use a cross-linking agent such as N,N'-methylenebisacrylamide (CAS: 110-26-9) to avoid cross-linking of the molecular chains into a network polymer, and the cross-linking agent A linear main chain is generated in the polymerized product under the condition that is not added. When the molecular chains cross-link to each other to form a network polymer, it forms a porous structure and affects the elution efficiency of the target protein.

In some preferred forms, the amount of acrylic acid used when manufacturing the magnetic microspheres B is 0.002-20 mol/L.

In some preferred forms, the amount of sodium acrylate used when manufacturing the magnetic microspheres C is 0.53-12.76 mol/L.

The outer surface of the biomagnetic microspheres can adopt other activation modification schemes than amination. For example, the aminated biomagnetic microspheres (amino group-modified magnetic microspheres A) can be further reacted with acid anhydrides or other modifying molecules, thereby carboxylating the outer surface of the biomagnetic microspheres. Or to realize chemical modification of other activation methods.

The immobilized molecule is a small molecule immobilized by covalently bonding a polymer backbone to the outer surface of a magnetic bead. The immobilized small molecule is not particularly limited, one end of which is covalently bound to the outer surface of the magnetic bead and the other end is capable of initiating polymerization reactions including homopolymerization reactions or copolymerization reactions or polycondensation reactions, or other It suffices if the ends can be copolymerized with the ends of the linear main chain of the polymer.

The immobilized molecule may be drawn with only one polymeric linear backbone, or with two or more polymeric linear backbones, as long as it does not involve chain deposition and/or an increase in retention rate. may Preferably, one immobilizing molecule draws only one polymer molecule and draws only one polymer linear backbone.

In some preferred forms, the immobilized molecule may have only one polymer linear backbone pulled out, or two or more polymer A linear backbone may be drawn. Preferably, one immobilizing molecule draws only one polymer molecule and draws only one polymer linear backbone.

In some other preferred embodiments, the acrylic monomer molecule, which is a monomer unit for polymerization, is acrylic acid, acrylate, acrylic acid ester, methacrylic acid, methacrylate, methacrylic acid ester monomer, or any of them. may be a combination of

As another embodiment of the present invention, the acrylic polymer can be replaced with other polymers. Criteria for selection were that the polymer formed had a single linear backbone, distributed a large number of side branches along the backbone, and carried functional groups on the side branches to facilitate subsequent chemical modification. to provide a large number of functional groups to the binding sites on the outer surface of the magnetic beads by means of branched chains distributed at the side ends of the polymeric linear backbone. Examples include ε-polylysine chains, α-polylysine chains, γ-polyglutamic acid chains, polyaspartic acid chains, aspartic acid/glutamic acid copolymers, and the like.

Methods for introducing polymer molecules of other alternative forms of the above polymers to the outer surface of the biomagnetic microspheres are described below. Based on the chemical structure of the polymer substitute and the type of its side-branched active groups, select the appropriate biomagnetic microsphere outer surface activation modification method, immobilized molecule type, monomer molecule type, A reaction is performed to introduce a large number of branched chain-located active groups to the outer surface of the biomagnetic microspheres.

After covalently bonding acrylic polymer molecules (e.g., sodium polyacrylate linear chains) to the outer surface of the magnetic beads, functional groups at the ends of the branched chains provide activation sites, or biotin or biotin analogue molecules are attached. Prior to ligation, the branched functional groups of the polymer molecule can be activated as required for the reaction, rendering it reactively active and forming activated sites. 3-Propanediamine is covalently attached to the active site of the polymer branch chain (each monomer acrylic unit structure can provide one active site) to form a new functional group (amino group) followed by carboxyl A covalent amidation reaction between a group and an amino group is used to covalently attach a biotin or biotin analogue molecule to a new functional group at the branched end of the polymer, and the biotin or biotin analogue is attached to the branched chain end of the polymer. Complete covalent bonding. As an example of using biotin as a purification medium, biotin-modified biomagnetic microspheres D can be obtained, one biotin molecule can provide one specific binding site. Take for example the functional group of the polymer branch chain is COONa, in this case sodium acrylate is used as the monomer molecule, which is first subjected to carboxyl group activation before covalent reaction with 1,3-propanediamine. A conventional carboxyl group activation method, such as adding EDC.HCl and NHS, can be used.

5.3.1. Preparation of acrylic polymer-modified magnetic microspheres Preparation of magnetic microsphere A: For aqueous solution of triiron tetroxide magnetic microspheres coated with silica, wash the magnetic microspheres with absolute ethanol and apply 3-aminopropyltriethoxysilane. An ethanol solution of (APTES, a coupling agent) is added, reacted, washed, and a large amount of amino groups introduced onto the outer surface of the magnetic microspheres.

Production of magnetic microsphere B: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) are added to an aqueous solution of acrylic acid to activate carboxyl groups. and added to the aqueous solution containing the magnetic microspheres A after activation. The activated carboxyl group in acrylic acid and the amino group on the outer surface of the magnetic microsphere form a covalent bond (amide bond), introducing a large amount of carbon-carbon double bonds to the outer surface of the magnetic microsphere.

Preparation of magnetic microsphere C: An aqueous solution of acrylic monomer molecules is added to magnetic microsphere B, an initiator is added, and a carbon-carbon double bond is polymerized. The carbon-carbon double bonds in the acrylic monomer molecules and the carbon-carbon double bonds on the surface of the magnetic microspheres undergo open bond polymerization, and the acrylic polymer molecules are covalently linked to the outer surface of the magnetic microspheres, where The acrylic polymer contains a carboxyl-based functional group, and the carboxyl-based functional group may exist in the form of a carboxyl group, a formate, a formate, or the like. In one preferred form, it is present in the form of sodium formate, in which case sodium acrylate or sodium methacrylate, for example, is the monomer molecule. In another preferred form, they are present in the form of formic acid esters, in which case, for example, acrylic or methacrylic acid esters are used as monomer molecules. Formates and formates can obtain relatively good reaction activity after being activated by carboxyl groups.

5.3.2. Preparation of Biotin-Modified Biomagnetic Microspheres D Solution of Magnetic Microspheres C: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) to prepare microspheres. After carboxyl-activating the carboxyl-based functional groups on the side branched chains of the polymer molecules on the outer surface of the sphere, an aqueous solution of propylenediamine is added to carry out a coupling reaction, and propylenediamine is added to the side-branched carboxyl groups of the acrylic polymer molecules. to convert the functional groups of the polymer side branches from carboxyl groups to amino groups.

Aqueous solution of biotin: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to activate the carboxyl groups in the biotin molecule, and then added to the aqueous solution containing magnetic microspheres C, Biotin is covalently coupled to the position of the new functional group (amino group) of the side branched chain of the polymer on the outer surface of the magnetic microsphere C, and the biotin molecule is linked to each of the many side branched chains of the acrylic polymer. Biomagnetic microspheres D are obtained.

5.3.3. Preferred Examples In some preferred embodiments, the method for producing the biomagnetic microspheres D is as follows.

First, weigh out 0.5-1000 mL (20%, v/v) of an aqueous solution of silica-coated triiron tetroxide magnetic microspheres, wash the magnetic microspheres with absolute ethanol, and add 10-300 mL of 3- An ethanol solution (5% to 50%, v/v) of aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the magnetic microspheres after the washing, reacted for 2 to 72 hours, and treated with absolute ethanol. and distilled water to obtain amino group-modified magnetic microspheres A.

Take 1.0×10 ⁻⁴ to 1 mol of acrylic acid and add it to solution X of pH 4 to 6 (solution X: final concentration of 0.01 to 1 mol/L of 2-morpholinoethanesulfonic acid (CAS: 4432-31- 9), 0.1-2 mol/L NaCl aqueous solution), 0.001-0.5 mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl, CAS: 25952-53- 8) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS, CAS: 6066-82-6) are added and allowed to react for 3-60 min. The above solution was added to a pH 7.2-7.5 PBS buffer solution mixed with 0.5-50 mL of magnetic microspheres A, reacted for 1-48 hours, washed the magnetic microspheres with distilled water, and carbon- Magnetic microspheres B modified with carbon double bonds are obtained.

Take 0.5-50 mL of magnetic microball B, add 0.5-200 mL of 5%-30% (w/v) sodium acrylate solution, and add 10 μL-20 mL of 2%-20% (w/v) ) Add an ammonium persulfate solution and 1 μL to 1 mL of tetramethylethylenediamine, react for 3 to 60 minutes, and then wash the magnetic microspheres with distilled water to obtain sodium polyacrylate-modified magnetic microspheres C.

Take 0.5-50 mL of magnetic microspheres C, transfer to pH 4-6 solution X, and add 0.001-0.5 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS) are added and allowed to react for 3-60 min. Subsequently, a PBS buffer solution of pH 7.2-7.5 in which 0.0001-1 mol of 1,3-propanediamine is dissolved is added and reacted for 1-48 hours. After washing with distilled water, a PBS buffer solution was added to convert the COONa of the polymer-side branched chain in the magnetic microsphere C into an amino functional group, and add 1.0×10 ⁻⁶ to 3.0×10 ⁻⁴ mol to the solution X. of biotin was weighed out and 2.0×10 ⁻⁶ to 1.5×10 ⁻³ mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0×10 ⁻⁶ to 1 Add 5×10 ⁻³ mol of N-hydroxysuccinimide and react for 3-60 min. Subsequently, it is added to the magnetic microsphere solution after washing, reacted for 1 to 48 hours, and washed with distilled water to obtain biotin-modified magnetic microsphere D.

5.4. A third aspect of the present invention provides biomagnetic microspheres obtained by reacting the biomagnetic microspheres provided in the first aspect of the present invention with avidin or an avidin analogue.

6. A sixth aspect of the present invention provides a method of manufacturing the biomagnetic microspheres provided in the second aspect of the present invention, the method comprising: (i) providing biomagnetic microspheres according to claim 1; which can be produced in steps (1) to (4) of the fifth aspect; and connecting to the body.

In some preferred forms, the biomagnetic microspheres are manufactured by the following steps. Steps (1) to (4) are the same as in the fifth aspect, and step (5) links the purification medium to biotin at the branched ends of the polymer of said biomagnetic microspheres.

7. A seventh aspect of the present invention provides a method of manufacturing the biomagnetic microspheres provided in the second aspect of the present invention, the method comprising: (i) providing biomagnetic microspheres according to claim 1; and (ii) forming a covalent complex of avidin or an avidin analogue with a purification medium to provide the purification medium. as a raw material for binding it to the branched ends of a polymer, forming an affinity complex binding action between said biotin or biotin analogue and said avidin or avidin analogue to produce a biomagnetic microsphere with a purification medium and obtaining.

independently optionally, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing;
Optionally, this can be accomplished independently by eluting the covalent complex of the avidin or avidin analogue with the purification medium, optionally including exchanging the purification medium under suitable conditions.

In some preferred forms, the biomagnetic microspheres are manufactured by the following five steps. Steps (1)-(4) are the same as in the fifth mode, and step (5) is the same as step (ii) above.

8. An eighth aspect of the invention provides a method of making biomagnetic microspheres provided in the fourth aspect of the invention.

A fourth aspect of the invention provides affinity protein magnetic microspheres.

The biomagnetic microspheres provided in the fourth aspect of the present invention are made from the biomagnetic microspheres provided in the first aspect, and further bind a covalent complex of avidin or an analogue thereof and an affinity protein. and the affinity complexes are carried on the polymer branches of the biomagnetic microspheres by the affinity complex action between biotin or its analogues and avidin or its analogues.

In one preferred form, the biomagnetic microspheres provided in the fourth aspect of the present invention are made from the biomagnetic microspheres provided in the first aspect of the present invention, and further comprise an avidin-affinity protein covalent complex It is obtained by combining E.

avidin-affinity protein covalent complex E: also called avidin-affinity protein complex E, a complex linked in a covalent manner, with avidin or an analogue thereof at one end and an affinity protein at the other end, Both are directly linked by a covalent bond or indirectly linked by a covalent linking element. The covalent linking groups include covalent bonds, linking peptides, and the like. Avidin-affinity protein complex E includes, for example, affinity proteins with streptavidin, where affinity proteins include but are not limited to protein A, protein G and/or protein L and the like. The avidin-affinity protein complex E further includes a streptavidin-protein A complex, a streptavidin-protein A fusion protein, and a streptavidin-enhanced green fluorescent protein-protein A fusion protein (Protein A-eGFP-Streptavidin ), Protein A-eGFP-Tamavidin2, Protein A-eGFP-Tamavidin1, etc., where eGFP broadly includes eGFP variants, Streptavidin is streptavidin, and both Tamavidin1 and Tamavidin2 are avidin analogs is.

Avidin specifically binds to biotin to form an affinity complex. The binding action of the affinity complex of avidin and biotin can be replaced by the binding action of another affinity complex, and similarly the effect of reusing the affinity protein after exchange can be realized. However, the affinity complexing action provided by avidin and biotin is more preferred. This is due to the high specificity and strong affinity between the two, and biotin adds to the binding domain with avidin, further increasing the binding carboxyl group by one, and avidin is easy to produce into affinity proteins and fusion proteins. It's for.

Criteria for selection of affinity conjugates: good specificity, strong affinity, and provide one chemically conjugable site by which they can be covalently attached to the ends of polymer branches, or after being chemically modified It can be covalently attached to the branched chain ends of the polymer.

8.1. Manufacturing process In some embodiments, the following step (5) is performed based on the biomagnetic microspheres (biotin magnetic microspheres) manufactured in the fifth aspect above to obtain magnetic microspheres using an affinity protein as a purification medium. get the system.

Step (5): Binding a covalent complex of avidin or an avidin analogue to an affinity protein (eg avidin-affinity protein covalent complex E). A specific binding interaction between biotin or an analogue thereof and avidin or an analogue thereof binds said covalent complex (e.g. avidin-affinity protein covalent complex E) to the branched end of the polymer, biotin or An affinity complex is formed between the analogue and avidin or an analogue thereof to form affinity protein magnetic microspheres.

For example, avidin-affinity protein covalent complex E (for example, an affinity protein with streptavidin, wherein the affinity protein is selected from, but not limited to, protein A, protein G or/and protein L, etc.) , added to a system of biotin-modified biomagnetic microspheres D, exploiting the extremely strong specific affinity between biotin and avidin (e.g., streptavidin) to non-covalently attach affinity proteins to polymer branched ends on the outer surface of magnetic beads. Affinity protein-modified magnetic beads are obtained that bind and can be used for the separation and purification of antibody-based substances, using the affinity protein as a purification medium and providing a binding site for capturing the target protein.

8.2. Example of Manufacturing Process The biomagnetic microspheres provided in the fourth aspect of the present invention are exemplified by being linked to the branched ends of the polymer in the manner of biotin-avidin-affinity protein, and are manufactured by the following steps: can do.

(1) The magnetic microsphere body is chemically modified, amino groups are introduced into the outer surface of the magnetic microsphere body to form amino group-modified magnetic microsphere A, and the magnetic microsphere body is coated with _SiO2 . magnetic material, the coupling agent is preferably an aminated silane coupling agent.

In one preferred form, the magnetic microsphere bodies are chemically modified using a coupling agent.

When the magnetic microsphere body is a magnetic material coated with SiO ₂ , a silane coupling agent can be used to chemically modify the magnetic microsphere body. At this time, the silane coupling agent is preferably an aminated silane coupling agent.

(2) covalent bonding of acrylic acid molecules to the outer surface of the magnetic microspheres A by utilizing the covalent bonding reaction between carboxyl groups and amino groups to introduce carbon-carbon double bonds; Magnetic microspheres B containing double bonds are formed.

(3) Polymerizing an acrylic monomer molecule (such as sodium acrylate) using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer chain is a linear main chain. and branched chains containing functional groups, the polymer is covalently bonded to the outer surface of magnetic microsphere B by one end of the linear backbone to form acrylic polymer-modified magnetic microsphere C.

A preferred embodiment of said functional group is a specific binding site.

Other preferred aspects of the functional group are consistent with the first aspect above.

(4) A biotin-modified biomagnetic microsphere D is obtained by covalently bonding biotin to the functional group contained in the branched chain of the polymer.

(5) binding the avidin-affinity protein covalent complex E to the branched end of the polymer through the specific binding action of biotin and avidin to form the binding action of the affinity complex between biotin and avidin; Affinity protein-modified biomagnetic microspheres are obtained (biomagnetic microspheres).

Optionally independently, (6) sedimenting the affinity protein magnetic microspheres with a magnet, removing the liquid phase, and washing.

Further independently optionally, (7) exchanging said avidin-affinity protein covalent complex E and eluting said avidin-affinity protein covalent complex E under suitable conditions. include.

8.3. Biomagnetic Microspheres: Avidin-Protein A conjugated Biomagnetic Microspheres F (Affinity Protein conjugated Biomagnetic Microspheres F, Protein A modified Biomagnetic Microspheres F, Protein A modified magnetic microspheres)

Biotin-modified biomagnetic microspheres D are added to the fusion protein solution of avidin-Protein A binding complex E (eg, Protein A-eGFP-Streptavidin, Protein A-eGFP-Tamvavidin2), mixed and incubated. The specific binding of avidin (eg, Streptavidin or Tamvavidin2) and biotin anchors Protein A to the terminal groups of polymer branches on the outer surface of biomagnetic microspheres D, resulting in avidin-protein A bound biomagnetic microspheres. Get Sphere F. In the resulting biomagnetic microsphere F structure, the acrylic acid polymer side-branches contain biotin-avidin-protein A affinity complex structures, covalently linked to the polymer's linear main chain branch points by the bio-ends. to form a non-covalent strong specific binding action of the affinity complex between biotin and avidin, between avidin and protein A is covalently linked, and between avidin and protein A Fluorescent labels can be inserted, and other connecting peptides can be inserted.

Avidin-Protein A fusion proteins such as Protein A-eGFP-Streptavidin fusion protein and Protein A-eGFP-Tamvavidin2 fusion protein can be obtained by in vitro cell-free protein synthesis by IVTT reaction. At this time, the biomagnetic microsphere D is mixed with the supernatant obtained after the IVTT reaction, and affinity Accomplish protein A binding.

8.4. The amount of affinity protein binding on the outer surface of the biomagnetic microspheres can be determined by the following method (using fluorescent protein eGFP labeling as an example).

First, after the binding reaction between the affinity protein solution and the magnetic beads is completed, the biomagnetic microspheres bound with the affinity protein are adsorbed with a magnet and sedimented. The liquid phase is then separated and collected and designated as flow-through. In this case the affinity protein concentration in the liquid phase is reduced. Calculating the fluorescence intensity of the fluorescent protein eGFP bound to the biomagnetic microspheres by measuring the change in the fluorescence value of the fluorescent protein eGFP in the supernatant obtained in the IVTT reaction before and after binding to the biomagnetic microspheres, Calculate the affinity protein concentration. Adsorption of biomagnetic microspheres to affinity proteins tends to saturate when the affinity protein concentration in the flow-through is essentially unchanged compared to the affinity protein concentration in the IVTT solution prior to incubation with the biomagnetic microspheres. , meaning that the fluorescence value of the corresponding fluorescent protein eGFP does not change significantly. A pure eGFP protein is taken to establish a standard curve of fluorescence values and eGFP protein concentration, thereby determining the content, concentration of avidin-affinity protein (eg: streptavidin-protein A) bound to the biomagnetic microspheres. was measured.

When antibodies are separated and purified using protein A-modified biomagnetic microspheres, the amount of antibody binding can be calculated by the following method (using bovine serum antibodies as an example). Incubate the protein A-modified magnetic beads with a solution of bovine serum antibodies (e.g., obtain by expressing antibodies using an in vitro protein synthesis system or obtain commercially), and after the reaction is complete, add an elution buffer. A solution is used to elute the bovine serum antibodies from the magnetic beads, and the separated bovine serum antibodies are present in the eluate. The concentration of bovine serum albumin in the eluate is determined using the Bradford method. At the same time, a microplate reader test is performed using BSA as a standard protein, the protein concentration of the purified antibody can be calculated using the standard protein as a reference, and the yield of separation and purification can be calculated.

8.5. Regeneration of biomagnetic microspheres: replacement of purification media (example with protein A as affinity protein)
Protein A elution and exchange: Elution of avidin simultaneously achieves synchronous desorption of protein A, hence avidin-protein A exchange.

For example, biomagnetic microspheres F modified with protein A are added with a denaturing buffer (containing urea and sodium dodecyl sulfate), incubated in a metal bath at 95° C., and avidin- which binds biotin in biomagnetic microspheres D. Protein A fusion protein (e.g., SPA-eGFP-Tamvavidin2) is eluted to obtain regenerated biomagnetic microspheres D (which release binding sites for biotin at the ends of polymer branched chains), followed by refolded biomagnetic microspheres. A solution containing a fresh avidin-protein A fusion protein (for example, the supernatant after the IVTT reaction of SPA-eGFP-Tamvavidin2) was added to the magnetic microsphere D, and the biotin-binding site of the released biomagnetic microsphere D was added. New avidin-Protein A (eg, SPA-eGFP-Tamvavidin2) is allowed to recombine, forming a new non-covalent specific binding interaction between biotin and avidin (eg, Tamvavidin2), thereby binding Protein A. The exchange is realized to obtain the regenerated biomagnetic microspheres F.

9. Magnetic Microsphere Position Control After obtaining the biomagnetic microspheres according to the first to fourth aspects of the invention (including but not limited to biomagnetic microsphere D and biomagnetic microsphere F), the magnet is It can be used to sediment the magnetic microspheres, remove the liquid phase, and wash away adsorbed miscellaneous proteins and/or other impurities.

By controlling the size of the magnetic microspheres and the chemical and structural parameters of the polymer, said magnetic microspheres can be stably suspended in a liquid phase and will not settle out within two days or even longer. Moreover, it can be stably suspended in a liquid system under the condition that stirring is not continued. Magnetic microspheres can be controlled to a nanosize of a few microns or even submicron, while the grafting density of the polymer on the outer surface of the magnetic microsphere can be adjusted, and furthermore the hydrophilicity, structural type, hydrodynamics of the polymer itself can be controlled. Characteristics such as radius, chain length, number of branches and length of branched chains can be adjusted, which allows the magnetic microsphere system to better control the suspension performance in the system, and the magnetic microsphere system and in vitro protein Provide sufficient contact with the synthesis reaction mixture system. One preferred magnetic microsphere size is about 1 micrometer.

10. A ninth aspect of the present invention provides use of the biomagnetic microspheres according to the first to fourth aspects of the present invention in separating and purifying proteinaceous substances.

In a preferred embodiment, the biomagnetic microspheres are used for separation and purification of antibody-based substances.

The definition of said antibody-based substance refers to the term part.

The present invention particularly provides the use of said biomagnetic microspheres in the separation and purification of antibodies, antibody fragments, antibody fusion proteins and antibody fragment fusion proteins.

When the purification medium is linked to the branched ends of the polymer by a linking element comprising an affinity complex, the use further comprises recycling the biomagnetic microspheres, ie reuse after exchange of the purification medium.

11. A tenth aspect of the present invention is the use of the biomagnetic microspheres according to the second or fourth aspect of the present invention in the separation and purification of antibody-based substances, particularly antibodies, antibody fragments, antibody fusion proteins, antibody fragments. Use in the isolation and purification of fusion proteins is provided.

Said purification medium is an affinity protein.

Preferably, said affinity protein is linked to the branched chain of the polymer in a biotin-avidin-affinity protein fashion.

When said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex (e.g. the biomagnetic microspheres according to the fourth aspect), said use further regenerates biomagnetic microspheres Includes use, ie reuse after exchange of the affinity protein.

In the biomagnetic microspheres of this use, the binding action of the affinity complex resides in the branched backbone between the affinity protein and the linear backbone of the polymer, at which time it is optionally reused after exchange of the affinity protein. and the biomagnetic microspheres can be regenerated and used. Preferably, the biotin-avidin affinity complex binding action resides in the branched backbone between the affinity protein and the linear backbone of the polymer, ie the affinity protein binds to the polymer branch in a biotin-avidin-affinity protein fashion. Once ligated, the biomagnetic microspheres can also be exchanged by avidin-avidin elution at this time.

12. An eleventh aspect of the present invention provides biomagnetic microspheres. At least one type of polymer having a linear main chain and a branched chain is provided on the outer surface of the magnetic microsphere main body, one end of the linear main chain is fixed to the outer surface of the magnetic microsphere main body, and The end is free from the outer surface of the main body of the magnetic microsphere, and a purification medium is linked to the branched chain end of the polymer of the magnetic microsphere, and the purification medium is an avidin-type tag, a polypeptide-type tag, or a protein-type tag. selected from tags, antibody-type tags, antigen-type tags, or combinations thereof.

In one preferred form, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof.

In one preferred form, the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

In a preferred embodiment, the polypeptide tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, and a tag containing a WRHPQFGG (SEQ ID No: 7) sequence. , a tag containing a variant sequence of WRHPQFGG (SEQ ID No: 7) , a tag containing a sequence of RKAAVSHW (SEQ ID No: 8) , a tag containing a variant sequence of RKAAVSHW (SEQ ID No: 8) , or a combination thereof or variants thereof, wherein the Streg tag includes WSHPQFEK (SEQ ID No: 9) and variants thereof.

In one preferred form, the protein-based tag is any tag selected from affinity proteins, SUMO tags, GST tags, MBP tags, and combinations thereof and variants thereof.

In one preferred form, the outer surface of the main body of the magnetic microsphere has at least one type of polymer having a linear main chain and a branched chain, and one end of the main linear chain is shared on the outer surface of the main body of the magnetic microsphere. Bindingly immobilized, the other end of the polymer is free from the outer surface of the main body of the magnetic microsphere, and the affinity protein is linked to the branched chain end of the polymer of said magnetic microsphere.

Preferably, there is also a binding function of the affinity complex on the branched backbone between said affinity protein and the linear backbone of the polymer.

More preferably, said affinity protein is selected from protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, or combinations thereof.

The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of Examples 2 to 6 below contains 9.78 mM pH 8.0 trishydroxymethylaminomethane hydrochloride (Tris-HCl), 80 mM acetic acid Potassium, 5 mM magnesium acetate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate; concentration is 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid , lysine, arginine and histidine, each amino acid concentration is 0.1 mM), 15 mM glucose, 320 mM maltodextrin (molar concentration is about 52 mg/mL with glucose unit calculation), 24 mM tripotassium phosphate , 2% (w/v) of polyethylene glycol 8000 (both final concentrations), and finally 50% by volume of cell extract (specifically yeast cell extract, more specifically Kluyveromyces - Add lactis cell extract).

wherein said Kluyveromyces lactis extract contains endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following manner. A modified strain based on Kluyveromyces lactis strain ATCC8585 was taken and a gene encoding T7 RNA polymerase was integrated into the genome of Kluyveromyces lactis by the method described in CN109423496A to obtain a modified strain, to which T7 RNA was added. The polymerase is expressed endogenously and the cell stock is cultured with the modified strain, followed by the production of cell extracts. The production process of Kluyveromyces lactis cell extract adopts prior art means, referring to the method described in CN109593656A. The manufacturing steps generally include the following. An appropriate amount of fermentation-cultured Kluyveromyces lactis cells can be provided as raw materials, the cells can be rapidly frozen with liquid nitrogen, the cells can be disrupted, and the supernatant can be collected by centrifugation to obtain a cell extract. The protein concentration in the obtained Kluyveromyces lactis cell extract is 20-40 mg/mL.

IVTT reaction: 15 ng/μL of DNA template (the encoded protein contains a fluorescent label) was added to the in vitro protein synthesis system, subjected to an in vitro protein synthesis reaction, uniformly mixed, and placed in a 25-30°C environment. Leave to react, reaction time is 6-18h. A protein encoded by the DNA template is synthesized to obtain an IVTT reaction containing the protein. The RFU value can be measured using the ultraviolet absorption method, and the concentration and standard curve of the RFU value can be combined to calculate the content of the protein.

Example 1 Preparation of biomagnetic microspheres (bound to biotin)
Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere bodies, magnetic beads, glass beads)
20 g of Fe ₃ O ₄ microspheres were put into a mixed solvent of 310 mL of ethanol and 125 mL of ethanol, 45 mL of 28% (wt) ammonia water was added, 22.5 mL of ethyl orthosilicate was added dropwise, and stirred at room temperature for 24 h. and washed with ethanol and water after the reaction. Using triiron tetroxide microspheres with different particle sizes (about 1 μm, 10 μm, 100 μm) as raw materials, the particle size of the resulting glass beads was controlled. Said different particle size triiron tetroxide microspheres can be produced by conventional technical means.

The resulting magnetic microspheres are also called magnetic microsphere bodies because they are used as substrates for modifying purification media and connecting elements-purification media.

The resulting magnetic microspheres have a magnetic core, can be position-controlled by the action of magnetic force, and can be moved, dispersed, sedimented, etc., and are therefore broadly defined as magnetic beads.

The resulting magnetic microspheres, which have a silica coating layer, are also called glass beads, and the magnetic core contains components or fractions such as polymers, purification media, components of in vitro protein synthesis systems, nucleic acid templates, and protein expression products. Adsorption to minutes can be reduced.

From the results of several experiments, it was found that when the particle size of the magnetic microspheres was about 1 μm, the ease of suspension, the persistence of suspension, and the binding efficiency to proteins were all the highest. The IVTT reaction solution is used to provide a mixed system of target proteins, and the binding efficiency to the target protein can be increased by more than 50% when the particle size of the magnetic microspheres is about 1 μm compared to when the particle size is 10 μm, It can be increased by more than 80% compared to a particle size of 10 μm.

Biotin magnetic beads were prepared with silica-coated magnetic microspheres by the following steps.

First, 50 mL of an aqueous solution of silica-coated triiron tetroxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) is weighed out, and the solid content is 20% (v/v). to sediment the magnetic microspheres, remove the liquid phase, and wash the magnetic microspheres with 60 mL of absolute ethanol each time for a total of 5 washes. An excess of 100 mL of an ethanol solution (25%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the magnetic microspheres after the above washing and incubated in a 50° C. water bath for 48 hours. mechanically stirred for 1 hour, then mechanically stirred in a water bath at 70 °C for 2 hours, sedimented the magnetic microspheres with a magnet, removed the liquid phase, and washed the magnetic microspheres with 60 mL of absolute ethanol each time, totaling The magnetic microspheres were washed twice, followed by washing the magnetic microspheres with 60 mL of distilled water each time, and the washing was repeated three times to obtain magnetic microspheres A.

Next, 0.01 mol of acrylic acid was taken and 100 mL of solution X (solution X: 2-morpholinoethanesulfonic acid (CAS: 4432-31-9) with a final concentration of 0.1 mol / L, 0.5 mol / L NaCl aqueous solution), 0.04 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04 mol of N-hydroxysuccinimide (CAS: 6066 -82-6), stirred at room temperature to mix homogeneously, stirred to react for 15 min, adjusted the pH of the solution to 7.2 with NaHCO ₃ solid powder, and added 10 mL of the above solution after pH adjustment. Add 100 mL of PBS buffer solution to which magnetic microsphere A was added, mechanically stir in a 30° C. water bath for 20 h, sediment the magnetic microspheres with a magnet, remove the liquid phase, and wash with 60 mL of distilled water each time. The magnetic microspheres were washed, and washing was repeated six times to obtain magnetic microspheres B.

Third, take 1 mL of magnetic microspheres B, add 12 mL of 15% (w/v) sodium acrylate solution, add 450 μL of 10% ammonium persulfate solution and 45 μL of tetramethylethylenediamine, and place at room temperature for 30 minutes. Allow to react, sediment the magnetic microspheres with a magnet, remove the liquid phase, wash the magnetic microspheres with 10 mL of distilled water each time for a total of 6 washes, and magnetic microspheres C (magnetic microspheres modified with acrylic polymer). Microspheres C) were obtained.

Fourth, transfer the synthesized magnetic microspheres C into 10 mL of solution X, add 0.004 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.004 mol of N-hydroxysuccinimide, Stir at room temperature to mix evenly, stir to react for 15 min, sediment the biomagnetic microspheres with a magnet, remove the liquid phase, wash 3 times with 10 mL distilled water each time, 4.0×10 ^{− 4} mol of 1,3-propanediamine was dissolved in 10 mL of PBS buffer solution, added to the washed magnetic microspheres, mechanically stirred in a 30° C. water bath for 20 hours, and sedimented the magnetic microspheres with a magnet; Remove the liquid phase, wash 6 times with 10 mL of distilled water each time, add 10 mL of PBS buffer solution, weigh 2.5×10 ⁻⁴ mol of biotin, add 10 mL of solution X, and add 1. Add 0×10 ⁻³ mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.001 mol of N-hydroxysuccinimide, stir uniformly at room temperature, stir and react for 15 min, add NaHCO ₃ Adjust the pH of the solution to 7.2 with solid powder, add it to the magnetic microspheres containing 10 mL of the PBS buffer solution washed above, mechanically stir in a 30° C. water bath for 20 hours, and remove the magnetic microspheres with a magnet. It was sedimented, the liquid phase was removed, and the biotin-modified biomagnetic microsphere D was obtained by washing ten times with 10 mL of distilled water each time.

Example 2 Preparation of Avidin-Protein A Binding Biomagnetic Microsphere F (Affinity Protein Binding Biomagnetic Microsphere F) (Protein A as Purification Medium and Biotin-Avidin Affinity Complex as Linking Element)

2.1 Synthesis of Protein A-eGFP-Avidin Fusion Protein DNA sequences composed of tripartite genes of fusion proteins Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were constructed respectively.

Here, Protein A is derived from Staphylococcus Aureus in sequence and abbreviated as SPA. The amino acid sequence of SPA has a total length of 516 amino acid residues, of which amino acids 37-327 were selected as the gene sequence used for fusion protein construction, ie, the antibody binding domain of SPA. After optimizing the sequence with an optimization program, the nucleotide sequence was obtained and identified by SEQ ID NO. : 1.

Tamvavidin2 is an avidin analogue, a protein with the ability to bind biotin. In 2009, Yamamoto et al. found that it has a very strong biotin affinity similar to streptavidin, and that it has better thermostability than streptavidin (2009_FEBS_Yamanomo T_Tamavidins -- novel avidin-like biotin-binding proteins from the Tamogitake mushroom, translation: novel avidin-like biotin-binding protein from Tamogitake).

The amino acid sequence of Tamavidin2 can be retrieved from relevant databases, for example UniProt B9A0T7, which contains a total of 141 amino acid residues, and the DNA sequence was obtained by codon conversion, optimization with an optimization program, and optimized Subsequent nucleotide sequences are for example SEQ ID NO. :2.

Further, the nucleotide sequence of the enhanced fluorescent protein eGFP according to this example is SEQ ID NO. :3 and is the A206K variant of eGFP, also called mEGFP.
Recombinant PCR method was adopted to construct DNA templates of two fusion proteins, Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, respectively. Subsequently, an in vitro cell-free protein synthesis method was adopted to obtain two fusion proteins, Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2, respectively. Adopting previously disclosed in vitro cell-free protein synthesis methods, refer to patent documents CN201610868691.6, WO2018161374A1, KR20190108180A, CN108535489B, etc. SPA-eGFP-Streptavidin and SPA-eGFP-Tamavidin2 were each expressed by the IVTT system. In general, the IVTT system includes cell extracts (including genome-integrated expression RNA polymerase), DNA templates, energy systems (for example, phosphocreatine-phosphocreatine kinase system), magnesium ions, sodium ions, components such as polyethylene glycol. was added and the reaction was carried out at 28-30°C. The reaction was terminated after 8-12 hours. The resulting IVTT reaction contained a Protein A-eGFP-avidin fusion protein corresponding to the DNA template.
IVTT reaction solutions of Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamvavidin2 were respectively obtained.

SPA-eGFP-Streptavidin (corresponding to "1" in FIG. 4) and SPA-eGFP-Tamavidin2 (corresponding to "2" in FIG. 4) were each expressed in the IVTT system, and the RFU value of the synthesized protein was measured, The results are shown in "total" in FIG.

The IVTT reaction solution of Protein A-eGFP-Streptavidin and Protein A-eGFP-Tamavidin2 was centrifuged at 4000 rpm and 4° C. for 10 minutes, and the supernatant was retained. Labeled as IVTT supernatant.

2.2 Production of Biomagnetic Microspheres F Bound to Protein A After incubation with the magnetic microsphere D for 1 hour, the content of the fusion protein in the biomagnetic microsphere F was measured according to the method described above, and the strength of the binding ability of the two fusion proteins was compared. The results are shown in Figure 4 "supernatant".

30 μL of 10% (v/v) biomagnetic microsphere D suspension was pipetted into binding/washing buffer (10 mM Na ₂ HPO ₄ , pH 7.4, 2 mM KH ₂ PO ₄ , 140 mM NaCl, 2.6 mM KCl) was washed three times.

Take 2 mL of the IVTT supernatant containing the avidin-Protein A fusion protein, rotate and incubate with the biomagnetic microspheres D described above at 4° C. for 1 hour, and collect the unbound supernatant, i.e. the flow-through. , this step was repeated three times to obtain three different flow-throughs. That is, the same lot of Biomagnetic Microsphere D was incubated with Avidin-Protein A three times consecutively. 2 mL of the IVTT supernatant described in step 2.1 was used each time, and the corresponding flow-through obtained each time was numbered 1, 2, 3 according to the order. Fluorometric method was adopted to detect the eGFP fluorescence value of the IVTT supernatant and the three times flow-through, the results are shown in Figure 5, the excitation wavelength (Ex) is 488 nm and the emission wavelength (Em) is 507 nm. is. The close RFU values of the triplicate flow-through to the RFU values of the IVTT supernatants explain that the binding of Biomagnetic Microspheres D to Avidin-Affinity Protein A tends to saturate, and the corresponding RFU values are shown in the table below. 1.

The RFU values for SPA-eGFP-Streptavidin and SPA-eGFP-Tamvavidin2 bound to Biomagnetic Microsphere D are shown in Tables 2 and 3 below, respectively. Here, for Streptavidin, the first binding already saturates the Protein A bound amount of the biomagnetic microsphere D. After the second binding to Tamvavidin2, the amount of Protein A binding of Biomagnetic Microsphere D almost reaches saturation. The first binding amount was calculated by subtracting the protein amount in flow-through 1 from the protein amount in the supernatant, and the second binding amount was calculated by subtracting the protein amount in flow-through 2 from the supernatant protein amount. The third binding amount was calculated by subtracting the protein amount in flow-through 3 from the protein amount in the supernatant, and is shown in Tables 2 and 3 below. Here, avidity refers to Protein A fusion protein/Biomagnetic Microsphere D and is the mass-to-volume ratio in units of (mg/mL).

Calculate the protein concentration bound each time based on the standard curve of fluorescence values and protein mass concentrations, and calculate the total protein amount bound each time based on the incubation volume (2 mL).
The formula for converting the estimated RFU value of eGFP to protein concentration based on a standard curve prepared from purified eGFP is as follows.

where X is the protein mass concentration (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), N is the molecular weight of SPA-eGFP-Streptavidin (77 .3 kDa) or the molecular weight of SPA-eGFP-Tamvavidin2 (79.4 kDa).

By substituting the measured RFU fluorescence value, the mass concentration (μg/mL) of the target protein SPA-eGFP-Streptavidin or SPA-eGFP-Tamvavidin2 can be calculated.

where Y is the RFU value of the IVV supernatant, flow-through of the avidin-Protein A fusion protein; The volume of the A solution can be multiplied to give the total protein amount of the avidin-Protein A fusion protein. Subtract the amount of avidin-protein A protein in the flow-through from the amount of avidin-protein A protein in the IVTT supernatant and the resulting difference is the amount of avidin-protein A protein bound to the biomagnetic microspheres W is. W was divided by the column bed volume of the magnetic microspheres to calculate the mass of avidin-Protein A fusion protein bound to the magnetic microspheres per unit volume, ie binding force, in mg/mL.

Since 30 μL of 10% (v/v) biomagnetic microsphere D suspension was used in this example, Tables 2 and 3 were converted into binding amounts of 1 mL of 100% biomagnetic microsphere D. shown in Here, the binding force between Tamvavidin2 and biomagnetic microsphere D is slightly stronger than that of Streptavidin.

Example 3 Purification of Serum Antibodies with Biomagnetic Microspheres F Modified with Protein A Biomagnetic microspheres bound with different volumes of SPA-eGFP-Streptavidin fusion protein were rotary incubated with 1 mL of newborn bovine serum for 1 hour at 4°C. followed by 3 washes with 1 mL binding/wash buffer, each time rotating at 4° C. for 10 minutes. The antibody was eluted with 100 μL of 0.1 M pH 2.8 glycine, the eluted antibody was in the eluate, and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was added to the eluate. added immediately. A Protein A agarose column manufactured by Seiko was used as a control (catalog number C600957-0005).

40 μL of the eluate was taken, 10 μL of 5×SDS sample buffer (containing no reducing agent) was added, subjected to a metal bath at 95° C. for 10 minutes, and subjected to SDS-PAGE electrophoresis detection. The detection results are shown in FIG. .

As can be seen from FIG. 6, a 6 μl column bed volume of biomagnetic microspheres bound with SPA-eGFP-Streptavidin fusion protein can be used to obtain better separation and purification effect than commercial products.

Washing the eluted biomagnetic microspheres three times with 1 mL of binding/washing buffer and repeating the incubation with antibody, washing and elution steps described above, i.e. after the first antibody incubation, washing and eluting; Bound antibody is released, protein A-modified magnetic microspheres are obtained again, incubation with antibody again, washing, elution are performed, and protein A-modified magnetic microspheres are obtained again, followed by a third cycle. Incubate, wash, and elute at After a total of 3 incubations of the biomagnetic microspheres with the antibody, 40 μL of the antibody-containing eluate obtained after the 3rd incubation with the antibody was taken and 10 μL of 5× sample buffer (without reducing agent) was added, After a metal bath at 95° C. for 10 minutes, SDS-PAGE electrophoresis detection was performed, and the detection results are shown in FIG.

As can be seen from FIGS. 6 and 7, the SPA-eGFP-Streptavidin-bound biomagnetic microspheres F can be repeatedly utilized multiple times, and after incubation with antibody and elution of antibody, a second, three Incubated with bovine serum again for the second time, both can extract antibodies in bovine serum, and the overall effect is not inferior to Protein A agarose magnetic beads manufactured by Seiko. For a column bed volume of 6 μL, the present invention has even higher efficiency. The biomagnetic microsphere F bound with the affinity protein (here, protein A) provided in the present invention can be used repeatedly multiple times.

Example 4 Recyclability of Binding of Biomagnetic Microsphere D and Affinity Complex E (Avidin-Protein A)

4.1 Incubation Incubation preparation of biomagnetic microspheres F conjugated with SPA-eGFP-Tamvavidin2:
Take 50 μL of 10% Biomagnetic Microsphere D suspension (volume is 5 μL) and wash 3 times with 2 mL binding/washing buffer, then add 2 mL of IVTT supernatant of SPA-eGFP-Tamvavidin2 at 4°C. 1 h at 4° C., discarded supernatant (supernatant was collected to give flow-through 1), repeated addition of fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2, and rotatable incubation at 4° C. for 1 h. discard the supernatant (collect the supernatant to give flow-through 2), add fresh IVTT supernatant containing SPA-eGFP-Tamvavidin2 for the third time, rotate and incubate at 4°C for 1 hour, The supernatant was discarded (supernatant was collected to give flow-through 3). The RFU value of the IVTT supernatant of SPA-eGFP-Tamvavidin2 and the flow-through of three times was measured, and the remaining content of SPA-eGFP-Tamvavidin2 in the solution was calculated based on the above formula, and the results are shown in FIG. 8 and Table 4. shown.

The formula for converting the estimated RFU value of eGFP to protein concentration based on a standard curve prepared from purified eGFP is as follows.

where X is the protein mass concentration (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), N is the molecular weight of SPA-eGFP-Tamvavidin2 (79 .4 kDa).

By substituting the measured RFU fluorescence value into the formula, the mass concentration (μg/mL) of the target protein SPA-eGFP-Tamvavidin2 can be obtained. For example, if the Y value is 1330.3, the corresponding X value is calculated to be 247.6 according to the above formula and is shown in Table 4.

4.2 Elution and Exchange Avidin-affinity protein complex E: SPA-eGFP-Tamvavidin2 was eluted and exchanged.

After washing the Biomagnetic Microspheres F three times with 2 mL binding/washing buffer, add 200 μL denaturing buffer (1 M Urea and 10% SDS) and place in a metal bath at 95° C. for 10 minutes to incubate. Avidin-Protein A bound to the biomagnetic microspheres was eluted. 20 μL of this eluate was taken, 5 μL of 5×SDS sample buffer was added, and SDS-PAGE electrophoresis was performed to obtain a band in lane 1 as shown in FIG. The eluted biomagnetic microspheres D were washed three times with 2 mL of binding/washing buffer to obtain the first renatured biomagnetic microspheres D, and the binding sites for biotin on the polymer branched chains on the outer surface of the magnetic microspheres were Released.

4.3 Second incubation (regeneration)
The SPA-eGFP-Tamvavidin2 incubation and the avidin-protein A elution process were repeated to achieve the first regeneration of the biomagnetic microspheres F, ie the avidin-protein A exchange for the first time. Biomagnetic Microsphere D regenerated in the first round bound to SPA-eGFP-Tamvavidin2 and the RFU values of the corresponding IVTT supernatants and triplicate flow-throughs were measured to obtain the data in Table 5. The eluted biomagnetic microsphere-D was washed with 2 mL of binding/washing buffer three times to obtain biomagnetic microsphere-D regenerated for the twelfth time.

4.4 Third Incubation (Second Regeneration)
The SPA-eGFP-Tamvavidin2 incubation and the avidin-protein A elution process were repeated again to achieve the second regeneration of the biomagnetic microspheres F, ie the avidin-protein A exchange for the second time. Biomagnetic Microsphere D, regenerated for the second time, bound SPA-eGFP-Tamvavidin2 and measured the RFU values of the corresponding IVTT supernatant and duplicate flow-throughs, yielding the data in Table 6. In Tables 4-6, avidity refers to Protein A fusion protein/Biomagnetic Microspheres D and is the mass-to-volume ratio in units (mg/mL).

The ability of renatured biomagnetic microspheres D to rebind with protein A-eGFP-avidin was measured.

Biomagnetic microsphere D obtained for the first time saturated-bound SPA-eGFP-Tamvavidin2 in the first time, and biomagnetic microsphere D regenerated in the first time saturated-bound SPA-eGFP-Tamvavidin2 in the second time. The biomagnetic microspheres D regenerated into SPA-eGFP-Tamvavidin2 were saturated bound to SPA-eGFP-Tamvavidin2 for the third time, protein was eluted with denaturation buffer, and three eluates each containing SPA-eGFP-Tamvavidin2 were collected. , SDS-PAGE was performed. The results are shown in FIG. Lanes 1, 2, 3 are biomagnetic microspheres F obtained for the first time after saturation binding of Protein A-eGFP-avidin in the first, second and third rounds, respectively, and biomagnetic microspheres F regenerated in the first round, respectively. Sphere F, representing the electrophoretic bands of the three elutions containing SPA-eGFP-Tamvavidin2 obtained by eluting proteins from the second renatured biomagnetic microsphere F;

4.5 Measurement of Bovine Serum Antibody Binding Capacity of Regenerated Biomagnetic Microspheres F Biomagnetic microspheres regenerated for the second time after saturation binding of biomagnetic microspheres D with SPA-eGFP-Tamvavidin2 for the third time. got an F. 2 mL of newborn bovine serum (commercially available, same as below) was added to the obtained biomagnetic microspheres F regenerated for the second time, and incubated with rotation at 4° C. for 1 hour, and the supernatant was discarded (supernatant was collected to obtain flow-through 1), 2 mL of newborn bovine serum was added again, incubated with rotation at 4° C. for 1 hour, and the supernatant was discarded (supernatant was collected to obtain flow-through 2). Washed 3 times with 1 mL binding/wash buffer, rotating incubation for 10 minutes at 4°C each time. Bovine serum antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine solution (antibodies bound to protein A ends on the magnetic microspheres) and 1/10 volume (i.e. 10 μL) of 1M was added to the eluted bovine serum antibody solution. A pH 8.0 Tris-HCl solution was immediately added to obtain 110 μL of eluate.

40 μL of the eluate was taken, 10 μL of 5×SDS sample buffer (no reducing agent) was added, subjected to a metal bath at 95° C. for 10 minutes, and then subjected to SDS-PAGE electrophoresis detection. The detection results are shown in FIG. As can be seen from the experimental results in FIGS. 7, 9 and Table 6, biomagnetic microsphere D has reproducible binding with SPA-eGFP-Tamvavidin2, and biomagnetic microsphere F provided in the present invention Can be used reproducibly. Also, the step of adding the denaturing solution and heat-treating does not affect the binding efficiency of the biomagnetic microspheres.

Example 5 Preparation of Biomagnetic Microspheres G with Protein A Binding (Protein G as Purification Medium and Biotin-Avidin Affinity Complex as Linking Element)

5.1 Synthesis of ProteinG-eGFP-Avidin Fusion Protein (Avidin-Affinity Protein Covalent Complex E)
A ProteinG-eGFP-Tamvavidin2 fusion protein was synthesized using the method of Example 2, and an IVTT reaction solution containing the ProteinG-eGFP-Tamvavidin2 fusion protein and an IVTT supernatant were sequentially produced.

First, the DNA sequence of ProteinG-eGFP-Tamvavidin2 fusion protein containing the three-stage gene sequence was constructed. Here, the nucleotide sequences of the "eGFP" segment and the "Tamvavidin2" segment are the same as in Example 2, and have SEQ ID Nos. : 3, SEQ ID No. :2. Here, the nucleotide sequence of the "Protein G" segment (SEQ ID No.: 4) was selected from the gene sequence of its antibody binding site from Streptococcus sp. group G.

Recombinant PCR method was adopted to construct the DNA template of ProteinG-eGFP-Tamvavidin2 fusion protein. In vitro amplification was performed using the RCA method. A ProteinG-eGFP-Tamvavidin2 fusion protein was synthesized using the indicated in vitro cell-free protein synthesis method (specifically employing a Kluyveromyces lactis-based cell extract).

An IVTT reaction was performed to sequentially obtain an IVTT reaction solution containing the ProteinG-eGFP-Tamvavidin2 fusion protein and an IVTT supernatant.

5.2 Preparation of Protein G-conjugated Biomagnetic Microsphere G Na ₂ HPO ₄ , pH 7.4, 2 mM KH ₂ PO ₄ , 140 mM NaCl, 2.6 mM KCl) three times.

IVTT supernatant containing 2 mL of ProteinG-eGFP-Tamvavidin2 fusion protein was taken and incubated with the biomagnetic microsphere D at 4° C. for 1 hour by rotation. Remaining fusion protein not bound to microspheres was included, and this step was repeated three times, each time incubating fresh IVTT supernatant with Biomagnetic Microsphere D, resulting in three different flow-throughs. That is, the same lot of biomagnetic microsphere D was sequentially incubated three times with the ProteinG-eGFP-Tamvavidin2 fusion protein in the IVTT supernatant. Using 2 mL of the IVTT supernatant obtained in step 5.1 each time, the corresponding three obtained flow-throughs were labeled sequentially as flow-through 1 (FT1), flow-through 2 (FT2), flow-through 3 (FT3). . Fluorometric method was adopted to detect the eGFP fluorescence value (measured by RFU value) of the IVTT supernatant and the three times flow-through, and the results are shown in FIG.

The fact that the flow-through RFU values are close to the IVTT supernatant RFU values explains the tendency of biomagnetic microsphere D to saturate binding to the ProteinG-eGFP-Tamvavidin2 fusion protein, and the corresponding RFU values are shown in Table 7 below. shown in The first binding amount was calculated by subtracting the protein amount in flow-through 1 from the protein amount in the supernatant, and the second binding amount was calculated by subtracting the protein amount in flow-through 2 from the supernatant protein amount. The third binding amount was calculated by subtracting the protein amount in flow-through 3 from the protein amount in the supernatant. The concentration of the ProteinG-eGFP-Tamvavidin2 fusion protein was calculated using the method of Example 2 using the following formula.

where X is the mass concentration of ProteinG-eGFP-Tamvavidin2 fusion protein (μg/mL), Y is the RFU fluorescence index, M is the molecular weight of eGFP (26.7 kDa), and N is ProteinG-eGFP - is the molecular weight of the Tamvavidin2 fusion protein.

Here, the binding force is the mass of the Protein G fusion protein bound to the magnetic beads and the volume of the magnetic beads used.

By the process of incubating the biomagnetic microspheres D and the IVTT supernatant containing the Protein G fusion protein, biomagnetic microspheres G to which Protein G is bound by an affinity complex (biotin-avidin) linkage method are obtained. Also referred to as magnetic beads.

5.3. Protein G magnetic bead-purified serum antibody (targeting IgG antibody)
Protein G magnetic beads bound with different volumes of ProteinG-eGFP-Tamvavidin2 fusion protein were rotary incubated with 1 mL of newborn calf serum for 1 hour at 4° C., followed by washing with 1 mL of binding/washing buffer (three times). , rotated for 10 min each time at 4°C. Antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was added immediately into the eluate. FIG. 11 shows the results of measuring the IgG antibody purity in the eluate by SDS-PAGE. Quantitative analysis is performed based on grayscale values and the purity is greater than 95%.

Example 6 Production of biomagnetic microsphere H bound with nanoantibody anti-eGFP (using nanoantibody anti-eGFP as purification medium and biotin-avidin affinity complex as linking element)

6.1. Synthesis of antiEGFP-mScarlet-avidin fusion protein (antiEGFP-mScarlet-Tamvavidin2 fusion protein, fusion protein of nanoantibodies) Using the method of Example 2, an antiEGFP-mScarlet-Tamvavidin2 fusion protein was synthesized (abbreviated as antiEGFP fusion protein). , molecular weight 59 kDa), an IVTT reaction solution containing the antiEGFP-mScarlet-Tamvavidin2 fusion protein, and an IVTT supernatant were sequentially prepared.

First, the DNA sequence of the antiEGFP-mScarlet-Tamvavidin2 fusion protein containing the triple gene sequence was constructed.

Here, the nucleotide sequence of the "Tamvavidin2" segment is the same as in Example 2, SEQSEQ ID No. :2.

Here, antiEGFP has an amino acid sequence of, for example, SEQ ID No. : Nanobody shown in 5.

Here mScarlet is a bright red fluorescent protein and the corresponding nucleotide sequence is SEQ ID No. :6.

Recombinant PCR method was adopted to construct the DNA template of antiEGFP-mScarlet-Tamvavidin2 fusion protein. In vitro amplification was performed using the RCA method. The antiEGFP-mScarlet-Tamvavidin2 fusion protein was synthesized using the in vitro cell-free protein synthesis method indicated above.

An IVTT reaction was performed to sequentially obtain an IVTT reaction solution containing the antiEGFP-mScarlet-Tamvavidin2 fusion protein and an IVTT supernatant.

6.2 Preparation of Nanobody anti-eGFP-conjugated Biomagnetic Microspheres H Pipette 30 μL of 10% (v/v) Biomagnetic Microspheres D obtained in Example 1 and add binding/washing buffer ( 10 mM _Na2HPO4 , pH 7.4, ₂ mM _KH2PO4 , 140 mM NaCl, 2.6 mM KCl) was washed _three times.

Take 2 mL of IVTT supernatant containing antiEGFP-mScarlet-Tamvavidin2 fusion protein (RFU value 2400), rotate and incubate with the biomagnetic microsphere D at 4 ° C. for 1 hour, collect the supernatant, which is the flow-through (RFU value 1700). The flow-through contained residual fusion protein not bound to microspheres. The conditions for measuring the RFU value were an excitation wavelength (Ex) of 569 nm and an emission wavelength (Em) of 593 nm.

Through the process of incubating the biomagnetic microspheres D and the IVTT supernatant containing the antiEGFP fusion protein, the incubated magnetic beads are bound to the nanoantibody anti-eGFP by affinity complex (biotin-Tamvavidin2) linkage, It is written as biomagnetic microsphere H, and also written as antiEGFP magnetic beads (nanoantibody magnetic beads).

6.3. Measurement of loading amount of antiEGFP magnetic beads that binds to eGFP protein (eGFP is the target protein, excess eGFP)
30 μL of 10% (w/v) of this Example 6.2. The anti-EGFP magnetic beads prepared in were pipetted and washed three times with binding/wash buffer and ready for use.

2 mL of the eGFP protein IVTT reaction solution (the nucleotide sequence encoding eGFP in the DNA template is shown in SEQ ID No. 3) was measured, and the fluorescence value was measured and recorded as the total fluorescence value. This was mixed with 3 μL of the washed anti-EGFP magnetic beads and incubated with rotation for 1 hour. Based on the fluorescence value measurement results in Table 8, the eGFP formula was used to obtain the corresponding amount of eGFP protein, and the resulting antiEGFP magnetic beads carried an amount of 17.7 mg/mL (antiEGFP magnetic per milliliter). mass of eGFP protein bound to the beads).

6.4. Efficiency of purification of eGFP protein by antiEGFP magnetic beads (eGFP is the target protein, excess magnetic beads)
The antiEGFP magnetic beads prepared in this Example 6.2 were taken, washed three times with binding/wash buffer and ready for use.

1 mL of the eGFP protein IVTT reaction solution (the nucleotide sequence encoding eGFP in the DNA template is shown in SEQ ID No. 3) was taken, and the fluorescence value was measured and indicated as Total. This was mixed with an excess of antiEGFP magnetic beads, incubated with rotation for 1 hour, the supernatant was collected, marked as Flow-through, and its fluorescence value was measured. The magnetic beads were washed twice with 1 mL of binding/washing buffer, each time rotated and incubated at 4° C. for 10 minutes, and the resulting washes were labeled as Washing1 and Washing2, respectively, and the respective fluorescence values were measured. Incubated magnetic beads bound to the target protein eGFP. FIG. 12 and Table 9 show the measurement results of the above fluorescence values. The results show that the anti-eGFP magnetic beads obtained in this scheme can effectively bind eGFP protein and elute it. Based on the fluorescence values of the IVTT supernatant and flow-through, the binding efficiency of the antiEGFP magnetic beads to the target protein eGFP was calculated to be 98.2% after the 1 hour incubation.

Antibodies were eluted with 100 μL of 0.1 M pH 2.8 glycine eluate and one tenth volume (10 μL) of 1 M pH 8.0 Tris-HCl was immediately added into the eluate. The fluorescence value of the eluate was measured, described as Elution, and the purity was examined by SDS-PAGE. As shown in FIG. 13, the purity was about 95%.

In addition, what has been described above is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the above-described embodiments. A person skilled in the art can make various changes and modifications within the conceptual scope of the technical solution of the present invention or under the guidance and suggestion thereof, and any changes and modifications with equivalent technical effects made , are all within the protection scope of the present invention.
<Appendix>
The present disclosure includes the following aspects.
<Item 1>
A biomagnetic microsphere comprising a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body has at least one polymer having a linear backbone and a branched chain, and one end of the linear backbone is immobilized on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and biotin or a biotin analogue is linked to the branched end of the polymer of the biomagnetic microsphere. A biomagnetic microsphere characterized by comprising:
<Section 2>
Item 2. The biomagnetic microspheres according to Item 1, wherein the branched chain ends of the polymer are linked to a purification medium via a linking element, and the linking element contains the biotin or biotin analogue. .
<Section 3>
the purification medium contains an avidin-type tag, a polypeptide-type tag, a protein-type tag, an antibody-type tag, an antigen-type tag, or a combination thereof;
Preferably, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof,
More preferably, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin,
More preferably, said avidin is streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof,
Preferably, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, a tag containing a variant sequence of WRHPQFGG, any one tag selected from a tag containing an RKAAVSHW sequence, a tag containing a variant sequence of RKAAVSHW, and a combination thereof, or a variant thereof;
Preferably, the protein-type tag is an affinity protein, a SUMO tag, a GST tag, an MBP tag, or any one tag selected from a combination thereof, or a variant thereof. More preferably, the affinity protein is a protein 3. The biomagnetic microsphere of paragraph 2, wherein the biomagnetic microsphere is selected from A, Protein G, Protein L, Denatured Protein A, Denatured Protein G, Denatured Protein L, and combinations thereof.
<Section 4>
the purification medium is linked to the branched chain ends of the polymer by a linking element comprising an affinity complex;
Preferably, said biotin or biotin analogue is linked to avidin or an avidin analogue by affinity complexing, said purification medium is directly or indirectly linked to said avidin or avidin analogue,
More preferably, the purification medium is linked to biotin or a biotin analogue at the branched end of the polymer by an avidin-type tag-purification medium covalent complex, and affinity between the biotin or biotin analogue is established by the avidin-type tag. form the connecting elements of the complex,
More preferably, the purification medium forms a linking element of an affinity complex with biotin or a biotin analog at the branched chain end of the polymer through an avidin-purification medium covalent complex. 4. The biomagnetic microsphere according to any one of 3.
<Section 5>
wherein the purification media and the polymer branched ends and the linking scheme are covalent bonds, supramolecular interactions, linking elements, or a combination thereof;
preferably said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acyl hydrazone bond, a disulfide bond, or a combination thereof;
Preferably, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions and combinations thereof;
More preferably, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction. 5. Biomagnetic microspheres according to any one of items 2 to 4.
<Section 6>
further comprising avidin bound to said biotin or biotin analogue, wherein said biotin or biotin analogue forms a binding function of an affinity complex with said avidin, preferably said avidin is streptavidin , modified streptavidin, streptavidin analogs, or combinations thereof.
<Item 7>
7. The biomagnetic microsphere of paragraph 6, further comprising an affinity protein linked to said avidin or avidin analogue.
<Item 8>
The size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0 .6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm , 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm , 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 700 μm , 850 μm, 900 μm, 950 μm, 1000 μm, or a range between any two particle size scales, said diameter size being an average value;
Preferably, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm,
Preferably, the average diameter of said magnetic microsphere bodies is 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm with deviation is ±20%, more preferably ±10%.
<Item 9>
the linear backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone;
Preferably, the linear backbone of said polymer is a polyolefin backbone and is provided from an acrylic polymer backbone,
More preferably, the monomer unit of the acrylic polymer is any one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof. 9. The biomagnetic microsphere according to any one of items 1 to 8, wherein
<Item 10>
the branched chains of the polymer are covalently attached to biotin or biotin analogues by covalent bonds based on functional groups;
Preferably, the covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group, wherein the functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group. , a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups.
<Item 11>
11. Any one of Items 1 to 10, wherein the linear main chain of the polymer is covalently bonded to the outer surface of the magnetic microsphere body directly or indirectly via a linking group. 10. The biomagnetic microspheres according to paragraphs.
<Item 12>
The magnetic microsphere body is a magnetic material coated with _SiO2 ,
Preferably, said magnetic material is an iron oxide, an iron compound, an iron alloy, a cobalt compound, a cobalt alloy, a nickel compound, a nickel alloy, a manganese oxide, a manganese alloy, or a combination thereof;
More preferably, the magnetic material is Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi , FeNiMo, FeSi, FeAl, FeSiAl, BaO.6Fe ₂ O ₃ , SrO.6Fe ₂ O ₃ , PbO.6Fe ₂ O ₃ , GdO, or a combination thereof. 1. Biomagnetic microspheres according to claim 1.
<Item 13>
Item 1. A method for producing biomagnetic microspheres according to item 1,
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. is a step
the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) Under the condition that no cross-linking agent is added, polymerize acrylic monomer molecules by using the polymerization reaction of carbon-carbon double bonds, and the resulting acrylic polymer is a linear main chain and a branched chain containing functional groups. wherein the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic acid-based polymer modified magnetic microsphere C;
(4) covalently coupling biotin or biotin analogues to the branch ends of the polymer via functional groups contained in the branched chains of the polymer to obtain the biomagnetic microspheres. .
<Item 14>
A method for producing a biomagnetic microsphere according to any one of Items 2 to 5,
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. a step of causing
the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) without adding a cross-linking agent, by utilizing the polymerization reaction of carbon-carbon double bonds, the acrylic monomer molecules are polymerized, and the resulting acrylic polymer is branched containing a linear main chain and functional groups; the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic polymer-modified magnetic microsphere C;
(4) biomagnetic microspheres D modified with biotin or biotin analogues by covalently coupling biotin or biotin analogues to the branched ends of the polymer via functional groups contained in the branched chains of the polymer; a step of obtaining
(5) linking a purification medium to biotin or a biotin analogue at the branched ends of the polymer in the biomagnetic microspheres D to obtain the biomagnetic microspheres with the purification medium attached;
Preferably, a covalent complex of avidin or an avidin analogue and a purification medium is attached to the branched ends of the polymer, allowing binding action of the affinity complex between said biotin or biotin analogue and said avidin or avidin analogue. forming to obtain said biomagnetic microspheres with purification media;
independently optionally, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing;
independently optionally comprising exchanging said avidin or avidin analogue with a covalent complex of a purification medium.
<Item 15>
Item 8. A method for producing biomagnetic microspheres according to Item 7,
Preferably, the method for producing biomagnetic microspheres comprises:
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. wherein the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) without adding a cross-linking agent, by utilizing the polymerization reaction of carbon-carbon double bonds, the acrylic monomer molecules are polymerized, and the resulting acrylic polymer is branched containing a linear main chain and functional groups; the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic polymer-modified magnetic microsphere C;
(4) covalently coupling biotin to the branched chain ends of the polymer through functional groups contained in the branched chains of the polymer to obtain biotin-modified biomagnetic microspheres D;
(5) linking the avidin-affinity protein covalent complex E to the branched chain end of the polymer, forming the binding action of the affinity complex between biotin and avidin, and the biomagnetic microparticles bound with the affinity protein; obtaining a sphere; and
independently and optionally, (6) sedimenting said biomagnetic microspheres with a magnet, removing the liquid phase and washing;
independently optionally comprising exchange of said avidin-affinity protein covalent complex E.
<Item 16>
Use of the biomagnetic microspheres according to any one of Items 1 to 12 in the separation and purification of proteinaceous substances,
Preferably, the biomagnetic microspheres are used in the separation and purification of antibody-based substances,
Use wherein said use optionally further comprises regenerating use of biomagnetic microspheres when said purification medium is linked to the branched ends of said polymer by a linking element comprising an affinity complex.
<Item 17>
Use of the biomagnetic microspheres according to any one of Items 2 to 5 or 7 for separation and purification of an antibody-based substance, wherein the purification medium is an affinity protein,
Preferably, said affinity protein is linked to the branched chains of the polymer in the manner of biotin-avidin-affinity protein,
Preferably, the antibody-based substance includes an antibody, an antibody fragment, an antibody fusion protein, an antibody fragment fusion protein,
When said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex, said use optionally further comprises a regeneration use of the biomagnetic microspheres, i.e. regeneration after exchange of the affinity protein. A use characterized by including utilization.
<Item 18>
1. A biomagnetic microsphere comprising a magnetic microsphere body, having at least one polymer having a linear backbone and a branched chain on the outer surface of the magnetic microsphere body, One end is fixed to the outer surface of the main body of the magnetic microsphere, the other end of the polymer is free from the outer surface of the main body of the magnetic microsphere, biotin is linked to the branched chain end of the polymer of the magnetic microsphere, and A biomagnetic microsphere characterized in that it is linked to avidin by the binding action of an affinity complex.
<Item 19>
A biomagnetic microsphere comprising a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body has at least one polymer having a linear backbone and a branched chain, and one end of the linear backbone is immobilized on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the branched chain end of the polymer of the magnetic microsphere is linked to the purification medium, and the the purification medium is selected from avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, and combinations thereof;
Preferably, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof,
More preferably, said avidin is streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof,
Preferably, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, a tag containing a variant sequence of WRHPQFGG, selected from any one of a tag comprising a RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof or variants thereof, wherein the Streg tag comprises WSHPQFEK and variants thereof;
Preferably, the protein-type tag is an affinity protein, a SUMO tag, a GST tag, an MBP tag, or any one tag selected from a combination thereof, or a variant thereof. More preferably, the affinity protein is a protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, and combinations thereof or variants thereof;
More preferably, the outer surface of the magnetic microsphere body has at least one polymer having a linear main chain and a branched chain, and one end of the linear main chain is shared with the outer surface of the magnetic microsphere body. The other end of the polymer is fixed by binding, the other end of the polymer is released from the outer surface of the main body of the magnetic microsphere, and the branched chain end of the polymer of the magnetic microsphere is linked to an affinity protein, more preferably the affinity protein. and the linear backbone of the polymer, more preferably said affinity protein is protein A, protein G, protein L, denatured protein A, denatured protein G , denatured Protein L, and combinations thereof.

Claims (19)

A biomagnetic microsphere comprising a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body has at least one polymer having a linear backbone and a branched chain, and one end of the linear backbone is immobilized on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and biotin or a biotin analogue is linked to the branched end of the polymer of the biomagnetic microsphere. A biomagnetic microsphere characterized by comprising: 2. The biomagnetic microstructure according to claim 1, wherein the branched ends of the polymer are linked to a purification medium via a linking element, and the linking element contains the biotin or biotin analogue. sphere. the purification medium contains an avidin-type tag, a polypeptide-type tag, a protein-type tag, an antibody-type tag, an antigen-type tag, or a combination thereof;
Preferably, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof,
More preferably, biotin is linked to the branched ends of the polymers of the biomagnetic microspheres, the purification medium is avidin, and an affinity complex is formed with the avidin,
More preferably, said avidin is streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof,
Preferably, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, a tag containing a variant sequence of WRHPQFGG, any one tag selected from a tag containing an RKAAVSHW sequence, a tag containing a variant sequence of RKAAVSHW, and a combination thereof, or a variant thereof;
Preferably, the protein-type tag is an affinity protein, a SUMO tag, a GST tag, an MBP tag, or any one tag selected from a combination thereof, or a variant thereof. More preferably, the affinity protein is a protein 3. The biomagnetic microspheres of claim 2, selected from A, Protein G, Protein L, Denatured Protein A, Denatured Protein G, Denatured Protein L, and combinations thereof. the purification medium is linked to the branched chain ends of the polymer by a linking element comprising an affinity complex;
Preferably, said biotin or biotin analogue is linked to avidin or an avidin analogue by affinity complexing, said purification medium is directly or indirectly linked to said avidin or avidin analogue,
More preferably, the purification medium is linked to biotin or a biotin analogue at the branched end of the polymer by an avidin-type tag-purification medium covalent complex, and affinity between biotin or a biotin analogue is established by an avidin-type tag. form the connecting elements of the complex,
More preferably, the purification medium forms a linking element of an affinity complex with biotin or a biotin analog at the branched chain end of the polymer through an avidin-purification medium covalent complex. 4. or the biomagnetic microsphere according to any one of 3. wherein the purification media and the polymer branched ends and the linking scheme are covalent bonds, supramolecular interactions, linking elements, or a combination thereof;
preferably said covalent bond is a dynamic covalent bond, more preferably said dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof;
Preferably, said supramolecular interactions are selected from coordinate bonds, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlap interactions, hydrophobic interactions and combinations thereof;
More preferably, said affinity complex interaction is selected from biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction. Biomagnetic microspheres according to any one of claims 2-4. further comprising avidin bound to said biotin or biotin analogue, wherein said biotin or biotin analogue forms a binding function of an affinity complex with said avidin, preferably said avidin is streptavidin , modified streptavidin, a streptavidin analogue, or a combination thereof. 7. The biomagnetic microsphere of Claim 6, further comprising an affinity protein linked to said avidin or avidin analogue. The size of the magnetic microsphere body is 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0 .6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm , 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm , 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 700 μm , 850 μm, 900 μm, 950 μm, 1000 μm, or a range between any two particle size scales, said diameter size being an average value;
Preferably, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm,
Preferably, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm,
Preferably, the average diameter of said magnetic microsphere bodies is 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm with deviation is ±20%, more preferably ±10%. the linear backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone;
Preferably, the linear backbone of said polymer is a polyolefin backbone and is provided from an acrylic polymer backbone,
More preferably, the monomer unit of the acrylic polymer is any one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof. The biomagnetic microspheres according to any one of claims 1 to 8. the branched chains of the polymer are covalently attached to biotin or biotin analogues by covalent bonds based on functional groups;
Preferably, the covalent bond based on the functional group means a covalent bond formed by participating in the covalent bond of the functional group, wherein the functional group is a carboxyl group, a hydroxyl group, an amino group, a mercapto group. , a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of said functional groups. 11. The linear backbone of the polymer is covalently bonded directly or indirectly via a linking group to the outer surface of the magnetic microsphere body. 2. The biomagnetic microsphere according to item 1. The magnetic microsphere body is a magnetic material coated with _SiO2 ,
Preferably, said magnetic material is an iron oxide, an iron compound, an iron alloy, a cobalt compound, a cobalt alloy, a nickel compound, a nickel alloy, a manganese oxide, a manganese alloy, or a combination thereof;
More preferably, the magnetic material is Fe ₃ O ₄ , γ-Fe ₂ O ₃ , iron nitride, Mn ₃ O ₄ , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi , FeNiMo, FeSi, FeAl, FeSiAl, BaO.6Fe ₂ O ₃ , SrO.6Fe ₂ O ₃ , PbO.6Fe ₂ O ₃ , GdO, or a combination thereof. A biomagnetic microsphere according to any one of claims 1 to 3. A method for producing biomagnetic microspheres according to claim 1, comprising:
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. is a step
the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) polymerizing acrylic monomer molecules using a carbon-carbon double bond polymerization reaction without adding a cross-linking agent, and the resulting acrylic polymer is a linear main chain and a branched chain containing functional groups; wherein the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic acid-based polymer modified magnetic microsphere C;
(4) covalently coupling biotin or biotin analogues to the branch ends of the polymer via functional groups contained in the branched chains of the polymer to obtain the biomagnetic microspheres. . A method for producing the biomagnetic microspheres according to any one of claims 2 to 5,
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. a step of causing
the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) without adding a cross-linking agent, by utilizing the polymerization reaction of carbon-carbon double bonds, the acrylic monomer molecules are polymerized, and the resulting acrylic polymer is branched containing a linear main chain and functional groups; the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic polymer-modified magnetic microsphere C;
(4) biomagnetic microspheres D modified with biotin or biotin analogues by covalently coupling biotin or biotin analogues to the branched ends of the polymer via functional groups contained in the branched chains of the polymer; a step of obtaining
(5) linking a purification medium to biotin or a biotin analogue at the branched ends of the polymer in the biomagnetic microspheres D to obtain the biomagnetic microspheres with the purification medium attached;
Preferably, a covalent complex of avidin or an avidin analogue and a purification medium is attached to the branched ends of the polymer, allowing binding action of the affinity complex between said biotin or biotin analogue and said avidin or avidin analogue. forming to obtain said biomagnetic microspheres with purification media;
independently optionally, (6) sedimenting the biomagnetic microspheres with a magnet, removing the liquid phase, and washing;
independently optionally comprising exchanging said avidin or avidin analogue with a covalent complex of a purification medium. A method for producing biomagnetic microspheres according to claim 7,
Preferably, the method for producing biomagnetic microspheres comprises:
(1) Chemically modifying the main body of the magnetic microsphere using an aminated silane coupling agent to introduce an amino group to the outer surface of the main body of the magnetic microsphere to form an amino group-modified magnetic microsphere A. wherein the magnetic microsphere body is a magnetic material coated with _SiO2 ;
(2) covalently coupling an acrylic acid molecule to the outer surface of the magnetic microsphere A using a covalent reaction between a carboxyl group and an amino group to introduce a carbon-carbon double bond; , forming carbon-carbon double bond-containing magnetic microspheres B;
(3) without adding a cross-linking agent, by utilizing the polymerization reaction of carbon-carbon double bonds, the acrylic monomer molecules are polymerized, and the resulting acrylic polymer is branched containing a linear main chain and functional groups; the polymer is covalently coupled to the outer surface of the magnetic microsphere B by one end of the linear backbone to form an acrylic polymer-modified magnetic microsphere C;
(4) covalently coupling biotin to the branched chain ends of the polymer through functional groups contained in the branched chains of the polymer to obtain biotin-modified biomagnetic microspheres D;
(5) linking the avidin-affinity protein covalent complex E to the branched chain end of the polymer, forming the binding action of the affinity complex between biotin and avidin, and the biomagnetic microparticles bound with the affinity protein; obtaining a sphere; and
independently and optionally, (6) sedimenting said biomagnetic microspheres with a magnet, removing the liquid phase and washing;
independently optionally comprising exchange of said avidin-affinity protein covalent complex E. Use of the biomagnetic microspheres according to any one of claims 1 to 12 in the separation and purification of proteinaceous substances,
Preferably, the biomagnetic microspheres are used in the separation and purification of antibody-based substances,
Use wherein said use optionally further comprises regenerating use of biomagnetic microspheres when said purification medium is linked to the branched ends of said polymer by a linking element comprising an affinity complex. Use of the biomagnetic microspheres according to any one of claims 2 to 5 or 7 in the separation and purification of an antibody-based substance, wherein the purification medium is an affinity protein,
Preferably, said affinity protein is linked to the branched chains of the polymer in the manner of biotin-avidin-affinity protein,
Preferably, the antibody-based substance includes an antibody, an antibody fragment, an antibody fusion protein, an antibody fragment fusion protein,
When said affinity protein is linked to the branched ends of said polymer by a linking element comprising an affinity complex, said use optionally further comprises a regeneration use of the biomagnetic microspheres, i.e. regeneration after exchange of the affinity protein. A use characterized by including utilization. 1. A biomagnetic microsphere comprising a magnetic microsphere body, having at least one polymer having a linear backbone and a branched chain on the outer surface of the magnetic microsphere body, One end is fixed to the outer surface of the main body of the magnetic microsphere, the other end of the polymer is free from the outer surface of the main body of the magnetic microsphere, biotin is linked to the branched chain end of the polymer of the magnetic microsphere, and A biomagnetic microsphere characterized in that it is linked to avidin by the binding action of an affinity complex. A biomagnetic microsphere comprising a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body has at least one polymer having a linear backbone and a branched chain, and one end of the linear backbone is immobilized on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the branched chain end of the polymer of the magnetic microsphere is linked to the purification medium, and the the purification medium is selected from avidin-type tags, polypeptide-type tags, protein-type tags, antibody-type tags, antigen-type tags, and combinations thereof;
Preferably, the avidin-type tag is avidin, an avidin analogue capable of binding to biotin, an avidin analogue capable of binding to a biotin analogue, or a combination thereof,
More preferably, said avidin is streptavidin, modified streptavidin, streptavidin analogues, or a combination thereof,
Preferably, the polypeptide-type tag is a CBP tag, a histidine tag, a C-Myc tag, a FLAG tag, a Spot tag, a C tag, an Avi tag, a Streg tag, a tag containing a WRHPQFGG sequence, a tag containing a variant sequence of WRHPQFGG, selected from any one of a tag comprising an RKAAVSHW sequence, a tag comprising a variant sequence of RKAAVSHW, or a combination thereof or variants thereof, wherein the Streg tag comprises WSHPQFEK and variants thereof;
Preferably, the protein-type tag is an affinity protein, a SUMO tag, a GST tag, an MBP tag, or any one tag selected from a combination thereof, or a variant thereof. More preferably, the affinity protein is a protein A, protein G, protein L, denatured protein A, denatured protein G, denatured protein L, and combinations thereof or variants thereof;
More preferably, the outer surface of the magnetic microsphere body has at least one polymer having a linear main chain and a branched chain, and one end of the linear main chain is shared with the outer surface of the magnetic microsphere body. The other end of the polymer is fixed by binding, the other end of the polymer is released from the outer surface of the main body of the magnetic microsphere, and the branched chain end of the polymer of the magnetic microsphere is linked to an affinity protein, more preferably the affinity protein. and the linear backbone of the polymer, more preferably said affinity protein is protein A, protein G, protein L, denatured protein A, denatured protein G , denatured Protein L, and combinations thereof.

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